APOLIPOPROTEIN C3 (APOC3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the apolipoprotein C3 (APOC3) gene, and methods of using such RNAi agents to inhibit expression of APOC3 and methods of treating subjects having an APOC3 associated disorder, e.g., hypertriglyceridemia.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/347,863, filed on Jun. 15, 2021, which is a continuation of U.S.patent application Ser. No. 17/128,320, filed on Dec. 21, 2020, now U.S.Pat. No. 11,034,958, issued on Jun. 15, 2021, which is a continuation ofU.S. patent application Ser. No. 16/534,193, filed on Aug. 7, 2019, nowU.S. Pat. No. 11,142,766, issued on Oct. 12, 2021, which is acontinuation application of U.S. patent application Ser. No. 15/597,225,filed on May 17, 2017, now U.S. Pat. No. 10,407,679, issued on Sep. 10,2019, which is a 35 § U.S.C. 111(a) continuation application whichclaims the benefit of priority to PCT/US2015/061065, filed on Nov. 17,2015, which claims priority to U.S. Provisional Application No.62/080,941, filed on Nov. 17, 2014, and U.S. Provisional Application No.62/136,159, filed on Mar. 20, 2015. The entire contents of each of theforegoing applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 27, 2022, isnamed 121301_02508_SL.txt and is 212,281 bytes in size.

BACKGROUND OF THE INVENTION

Apolipoprotein C3 (APOC3) is a very low density lipoprotein (VLDL) andan important regulator of lipoprotein metabolism. In humans, APOC2 isencoded by the APOC3 gene that is located in a gene cluster togetherwith the APOA1 and APOA4 genes on the long arm of chromosome 11. APOC3is expressed in the liver and, to a lesser extent, in the intestines, asa small 99-amino acid protein. Following removal of the 20-amino-acidsignal peptide in the endoplasmic reticulum, a mature ApoC3 protein of79 amino acids is formed, which may be present as a non-glycosylated ora glycosylated isoform.

The primary role of APOC3 is as a regulator of lipolysis throughnon-competitive inhibition of endothelial bound lipoprotein lipase(LPL). LPL hydrolyses triacylglycerols in triacylglycerol(triglyceride)-rich lipoproteins (TRLs), releasing fatty acids into theplasma and transforming large triacylglycerol-rich particles intosmaller triacylglycerol-depleted remnant lipoproteins. Individualslacking APOC3 have low TRL levels, coupled with highly efficientlipolysis of triacylglycerols. Furthermore, mice in which the APOC3 genehas been genetically deleted were also shown to have low plasmatriacylglycerol levels and efficient TRL catabolism. APOC3 also inhibitshepatic lipase (HL), a lipolytic enzyme with triacylglycerol lipase andphospholipase A1 activity that is synthesized in the liver. Theinhibitory effect of APOC3 on HL further reduces the lipolysis anduptake of TRL remnants by the liver. APOC3 has also been shown tostimulate synthesis of very low density lipoproteins (VLDLs). It isbelieved that the underlying mechanisms associated with this effect ofAPOC3 may relate to the inhibition of proteasome mediated degradation ofAPOB, resulting in increased APOB synthesis and secretion, and increasedsynthesis of VLDL triacylglycerols. APOC3 may, therefore, play a keyrole in regulating VLDL output by the liver.

Cellular studies report that APOC3 may interfere with TRL and remnantbinding to hepatic lipoprotein receptors. APOC3 can abolish APOB- andApoE-mediated binding of lipoproteins to low density lipoproteinreceptor (LDLR), either by masking or altering the conformation of APOBand APOE. The binding of chylomicrons and VLDL particles to thelipolysis-stimulated receptor (LSR) is also significantly inhibited byAPOC3.

An increase in APOC3 levels induces the development ofhypertriglyceridemia, or high (hyper-) blood levels (-emia) oftriglycerides. Elevated levels of triglycerides are associated with avariety of diseases, including cardiovascular disease, atherosclerosis,non-alcoholic fatty liver disease, non-alcoholic steatohepatitis,polycystic ovary syndrome, kidney disease, obesity, type 2 diabetesmellitus (insulin resistance), hypertension and skin lesions(xanthomas). Very high triglyceride levels also increase the risk ofacute pancreatitis. Therefore, regulating APOC3 metabolism may be animportant new therapeutic approach to managing hypertriglyceridemia andthe associated diseases.

Accordingly, there is a need in the art for regulators of APOC3expression for treating apolipoprotein C3 associated disorders, such ashypertriglyceridemia.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which inhibit or reducethe expression of APOC3 gene. The gene may be within a cell, e.g., acell within a subject, such as a human.

The present invention also provides methods and therapies for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an APOC3 gene, e.g., an apolipoprotein C3-associateddisease, such as hypertriglyceridemia, using iRNA compositions whichinhibit or reduce the expression of the APOC3 gene.

In some embodiments, the present invention provides a double strandedRNAi agent for inhibiting expression of apolipoprotein C3 (APOC3) in acell, wherein the double stranded RNAi agent comprises a sense strandand an antisense strand forming a double-stranded region, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1,and the antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:2,

wherein substantially all of the nucleotides of at least one strand aremodified nucleotides, and wherein said sense strand is conjugated to aligand attached at the 3′-terminus.

In certain aspects, all of the nucleotides of the sense strand and allof the nucleotides of the antisense strand are modified nucleotides. Inone aspect, the sense strand and the antisense strand comprise a regionof complementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the sequenceslisted in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

In some embodiments, at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, a nucleotide comprising a5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotidecomprising adenosine-glycol nucleic acid (GNA), a nucleotide comprisingthymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising2′-deoxythymidine-3′phosphate, a nucleotide comprising2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to acholesteryl derivative or a dodecanoic acid bisdecylamide group.

In one embodiment, substantially all of the nucleotides of the sensestrand are modified. In another aspect, substantially all of thenucleotides of the antisense strand are modified. In yet anotherembodiment, substantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides. In one embodiment, all of the nucleotides of thesense strand are modified nucleotides. In another embodiment, all of thenucleotides of the antisense strand are modified nucleotides. In yetanother embodiment, all of the nucleotides of the sense strand and allof the nucleotides of the antisense strand are modified nucleotides.

In one aspect, at least one strand comprises a 3′ overhang of at least 1nucleotide. In another aspect, at least one strand comprises a 3′overhang of at least 2 nucleotides.

In some embodiment, the present invention provides a double strandedRNAi agent capable of inhibiting the expression of apolipoprotein C3(APOC3) in a cell, wherein the double stranded RNAi agent comprises asense strand and an antisense strand forming a double stranded region,wherein the antisense strand comprises a region complementary to part ofan mRNA encoding APOC3, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

(III) sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(i)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 nucleotides which are either modified orunmodified or combinations thereof; each n_(p), n_(p)′, n_(q), andn_(q)′, each of which may or may not be present, independentlyrepresents an overhang nucleotide;

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and

wherein the sense strand is conjugated to at least one ligand.

In a further embodiment, i is 0; j is 0; i is 1; j is 1; both i and jare 0; or both i and j are 1. In another further embodiment, k is 0; 1is 0; k is 1; 1 is 1; both k and l are 0; or both k and 1 are 1. Inanother aspect, the YYY motif occurs at or near the cleavage site of thesense strand. In yet another aspect, the Y′Y′Y′ motif occurs at the 11,12 and 13 positions of the antisense strand from the 5′-end.

In one embodiment, Y′ is 2′-O-methyl or 2′-fluoro.

In some aspects, formula (III) is represented by formula (IIIa):

(IIIa) sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p′)-N_(a′)-Y′Y′Y′ N_(a′)-n_(q′) 5′.

In a further aspect, the double-stranded region is 15-30 nucleotidepairs in length. In another aspect, the double-stranded region is 17-23nucleotide pairs in length. In another embodiment, the double-strandedregion is 17-25 nucleotide pairs in length. In yet another embodiment,the double-stranded region is 23-27 nucleotide pairs in length. In afurther aspect, the double-stranded region is 19-21 nucleotide pairs inlength. In yet another aspect, the double-stranded region is 21-23nucleotide pairs in length.

In one embodiment, each strand has 15-30 nucleotides. In a furtherembodiment, each strand has 19-30 nucleotides.

In one aspect, the modifications on the nucleotides are selected fromthe group consisting of the modifications as listed in Tables 5, 9, 10,11B, 12, 13, and combinations thereof.

In some embodiments, the modifications on the nucleotides are2′-O-methyl and 2′-fluoro modifications.

In some embodiments, the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker. In a furtherembodiment, the ligand is

In some aspects, the ligand is attached to the 3′ end of the sensestrand.

In certain embodiments, the RNAi agent is conjugated to the ligand asshown in the following schematic

wherein X is O or S.

In some aspects, the RNAi agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage. In afurther aspect, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In anotherfurther aspect, the strand is the antisense strand. In yet anotheraspect, the strand is the sense strand.

In some embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In afurther aspect, the strand is the antisense strand. In another furtheraspect, the strand is the sense strand.

In certain embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In one embodiment, the strand is the antisense strand.

In some aspects, the RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In a further embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In some embodiments, the base pair at the 1 position of the 5′-end ofthe antisense strand of the duplex is an AU base pair.

In some aspects, the Y nucleotides contain a 2′-fluoro modification. Ina further aspect, the Y′ nucleotides contain a 2′-O-methyl modification.

In some embodiments, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In some aspects, the RNAi agent is selected from the group of RNAiagents listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B and 12.

In certain embodiments, the present invention also provides a doublestranded RNAi agent capable of inhibiting the expression ofapolipoprotein C3 (APOC3) in a cell, wherein the double stranded RNAiagent comprises a sense strand and an antisense strand forming adouble-stranded region,

wherein the sense strand comprises 5′-GCUUAAAAGGGACAGUAUUCU-3′ (SEQ IDNO: 13), and the antisense strand comprises5′-AGAAUACUGUCCCUUUUAAGCAA-3′ (SEQ ID NO: 14),

wherein substantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides,

wherein the sense strand is conjugated to a ligand attached at the3′-terminus, and

wherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.

In other embodiments, the present invention also provides a doublestranded RNAi agent capable of inhibiting the expression ofapolipoprotein C3 (APOC3) in a cell, wherein the double stranded RNAiagent comprises a sense strand and an antisense strand forming adouble-stranded region,

wherein the sense strand comprises 5′-GCUUAAAAGGGACAGUAUUCU-3′ (SEQ IDNO: 13), and the antisense strand comprises5′-UGAAUACUGUCCCUUUUAAGCAA-3′ (SEQ ID NO: 15),

wherein substantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides,

wherein the sense strand is conjugated to a ligand attached at the3′-terminus, and

wherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.

In certain embodiments, the present invention also provides a doublestranded RNAi agent capable of inhibiting the expression ofapolipoprotein C3 (APOC3) in a cell, wherein the double stranded RNAiagent comprises a sense strand and an antisense strand forming adouble-stranded region,

wherein the sense strand comprises 5′-GCUUAAAAGGGACAGUAUUCA-3′ (SEQ IDNO:659), and the antisense strand comprises5′-UGAAUACUGUCCCUUUUAAGCAA-3′ (SEQ ID NO:670),

wherein substantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides,

wherein the sense strand is conjugated to a ligand attached at the3′-terminus, and

wherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.

In embodiment, all of the nucleotides of the sense strand are modifiednucleotides. In one embodiment, all of the nucleotides of the antisensestrand are modified nucleotides. In another embodiment, all of thenucleotides of the sense strand and all of the nucleotides of theantisense strand are modified nucleotides.

In a further aspect, at least one of the modified nucleotides isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, a nucleotide comprising a5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotidecomprising adenosine-glycol nucleic acid (GNA), a nucleotide comprisingthymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising2′-deoxythymidine-3′phosphate, a nucleotide comprising2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to acholesteryl derivative or a dodecanoic acid bisdecylamide group.

In one embodiment, the RNAi agent comprises no more than 10 nucleotidescomprising 2′-fluoro modifications. In another embodiment, the RNAiagent comprises no more than 9 nucleotides comprising 2′-fluoromodifications. In another embodiment, the RNAi agent comprises no morethan 8 nucleotides comprising 2′-fluoro modifications. In anotherembodiment, the RNAi agent comprises no more than 7 nucleotidescomprising 2′-fluoro modifications. In another embodiment, the RNAiagent comprises no more than 6 nucleotides comprising 2′-fluoromodifications. In another embodiment, the RNAi agent comprises no morethan 5 nucleotides comprising 2′-fluoro modifications. In yet anotherembodiment, the sense strand comprises no more than 4 nucleotidescomprising 2′-fluoro modifications. In another embodiment, the sensestrand comprises no more than 4 nucleotides comprising 2′-fluoromodifications. In another embodiment, the sense strand comprises no morethan 3 nucleotides comprising 2′-fluoro modifications. In anotherembodiment, the sense strand comprises no more than 2 nucleotidescomprising 2′-fluoro modifications. In another aspect, the antisensestrand comprises no more than 6 nucleotides comprising 2′-fluoromodifications. In another embodiment, the antisense strand comprises nomore than 5 nucleotides comprising 2′-fluoro modifications. In anotherembodiment, the the antisense strand comprises no more than 4nucleotides comprising 2′-fluoro modifications. In another embodiment,the antisense strand comprises no more than 3 nucleotides comprising2′-fluoro modifications. In yet another aspect, the antisense strandcomprises no more than 2 nucleotides comprising 2′-fluoro modifications.

In one embodiment, the double-stranded RNAi agent of the inventionfurther comprises a 5′-phosphate or a 5′-phosphate mimic at the 5′nucleotide of the antisense strand. In another embodiment, thedouble-stranded RNAi agent further comprises a 5′-phosphate mimic at the5′ nucleotide of the antisense strand. In a specific embodiment, the5′-phosphate mimic is a 5′-vinyl phosphate (5′-VP).

In certain aspects, the ligand is

In some embodiments, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S.

In some aspects, the present invention provides a double stranded RNAiagent comprising the RNAi sequences listed in any one of Tables 4A, 4B,5, 8, 9, 10, 11A, 11B, 12, and 13.

In one embodiment, the RNAi agent is AD-57553 comprising the followingsequence:

sense: (SEQ ID NO: 16) 5′ GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3′antisense: (SEQ ID NO: 17) 5′ asGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsAfsa 3′.

In another embodiment, the RNAi agent is AD-65696 comprising thefollowing sequence:

sense: (SEQ ID NO: 18) 5′ GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3′antisense: (SEQ ID NO: 19) 5′ VPusGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsasa 3′.

In yet another aspect, the RNAi agent is AD-65703 comprising thefollowing sequence:

sense: (SEQ ID NO: 20) 5′ gscsuuaaAfaGfGfGfacaguauucaL96 3′ antisense:(SEQ ID NO: 21) 5′ usGfsaauAfcUfGfucccUfuUfuaagcsasa 3′.

In yet another aspect, the RNAi agent is AD-65704 comprising thefollowing sequence:

sense: (SEQ ID NO: 22) 5′ gscsuuaaAfaGfGfGfacaguauucaL96 3′ antisense:(SEQ ID NO: 23) 5′ usGfsaauacugucccUfuuuaagcsasa 3′.

In yet another aspect, the RNAi agent is AD-67221 comprising thefollowing sequence:

sense: (SEQ ID NO: 714) 5′ cscscaauAfaAfGfCfuggacaagaaL96 3′ antisense:(SEQ ID NO: 718) 5′ usUfscuuGfuCfCfagcuUfuAfuugggsasg 3′

In one embodiment, the RNAi agent is AD-69535 comprising the followingsequence:

sense: (SEQ ID NO:738) 5′ gscsuuaaaaGfgGfacaguauuca 3′ antisense:(SEQ ID NO:749) 5′ sGfsaauacugucCfcUfuuuaagcsasa 3′

In another embodiment, the RNAi agent is AD-69541 comprising thefollowing sequence:

sense: (SEQ ID NO:744) 5′ gscsuuaaaaGfgGfacagu(Agn)uuca 3′ antisense:(SEQ ID NO:755) 5′ usGfsaauacugucCfcUfuuuaagcsasa 3′

In certain embodiments, the present invention also provides acomposition comprising a modified antisense polynucleotide agent,wherein the agent is capable of inhibiting the expression of APOC3 in acell, and comprises a sequence complementary to a sense sequenceselected from the group of the sequences listed in any one of Tables 4A,4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, wherein the polynucleotide isabout 14 to about 30 nucleotides in length.

In some aspects, the present invention also provides a vector containingthe double stranded RNAi agent as described herein. In other aspects,the present invention also provides a cell containing the doublestranded RNAi agent as described herein.

In some embodiments, the present invention relates to a pharmaceuticalcomposition comprising the double stranded RNAi agent, or thecomposition comprising a modified antisense polynucleotide agent, or thevector as described herein.

In certain aspects, the double stranded RNAi agent is present in anunbuffered solution. In a further aspect, the unbuffered solution issaline or water. In other aspects, the double stranded RNAi agent ispresent in a buffered solution. In a further embodiment, the buffersolution comprises acetate, citrate, prolamine, carbonate, or phosphateor any combination thereof. In a specific embodiment, the buffersolution is phosphate buffered saline (PBS).

In one embodiment, the present invention also provides a method ofinhibiting apolipoprotein C3 (APOC3) expression in a cell, the methodcomprising:

-   -   (a) contacting the cell with the double stranded RNAi agent, or        the composition comprising a modified antisense polynucleotide        agent, the vector, or the pharmaceutical composition as        described herein; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of a        APOC3 gene, thereby inhibiting expression of the APOC3 gene in        the cell.

In one aspect, the cell is within a subject. In a further aspect, thesubject is a human or a rabbit. In one embodiment, the subject suffersfrom an APOC3 associated disease.

In some embodiments, the APOC3 expression is inhibited by at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 98% or about 100%.

In some aspects, the present invention provides a method of treating asubject having an apolipoprotein C3 (APOC3) associated disease,comprising administering to the subject a therapeutically effectiveamount of the double stranded RNAi agent, or the composition comprisinga modified antisense polynucleotide agent, or the vector, or thepharmaceutical composition as described herein, thereby treating saidsubject.

In one embodiment, the APOC3 associated disease is hypertriglyceridemia.In another embodiment, the APOC3 associated disease is selected from thegroup consisting of non-alcoholic fatty liver disease, non-alcoholicsteatohepatitis, polycystic ovary syndrome, kidney disease, obesity,type 2 diabetes mellitus (insulin resistance), hypertension,artherosclerosis and pancreatitis.

In some aspects, the double stranded RNAi agent is administered at adose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about50 mg/kg. In a further aspect, the double stranded RNAi agent isadministered at a dose of about 10 mg/kg to about 30 mg/kg. In anotheraspect, the double stranded RNAi agent is administered at a dose ofabout 3 mg/kg. In yet another aspect, the double stranded RNAi agent isadministered at a dose of about 10 mg/kg.

In one embodiment, the double stranded RNAi agent is administeredsubcutaneously. In another embodiment, the double stranded RNAi agent isadministered intravenously. In another embodiment, the double strandedRNAi agent is administered intramuscularly.

In some aspects, the RNAi agent is administered in two or more doses. Ina further aspect, the RNAi agent is administered at intervals selectedfrom the group consisting of once every about 12 hours, once every about24 hours, once every about 48 hours, once every about 72 hours, and onceevery about 96 hours.

In certain embodiments, the methods of the invention further compriseadministering to the subject an additional therapeutic agent. In afurther embodiment, the additional therapeutic agent is selected fromthe group consisting of an HMG-CoA reductase inhibitor, a fibrate, abile acid sequestrant, niacin, an antiplatelet agent, an angiotensinconverting enzyme inhibitor, an angiotensin II receptor antagonist, anacylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterolabsorption inhibitor, a cholesterol ester transfer protein (CETP)inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor,a cholesterol modulator, a bile acid modulator, a peroxisomeproliferation activated receptor (PPAR) agonist, a gene-based therapy, acomposite vascular protectant, a glycoprotein IIb/IIIa inhibitor,aspirin or an aspirin-like compound, an IBAT inhibitor, a squalenesynthase inhibitor, a monocyte chemoattractant protein (MCP)-Iinhibitor, or fish oil.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the relative amount of APOC3 mRNA in Hep3Bcells after treatment with a single dose of 0.1 nM or 10 mM of theindicated iRNAs of the invention.

FIG. 2 is a bar graph showing the relative amount of APOC3 mRNA measuredon day 5 in wild-type mice treated with 3, 10 and 30 mg/kg dose ofGalNac-conjugated AD-57558.

FIG. 3 is a bar graph showing the measured levels of APOC3 mRNA measuredin individual APOC3-AAV mice injected with AD-57553, AD-57547 andAD-58924.

FIG. 4 is a bar graph showing the group averages of the levels of APOC3mRNA measured in APOC3-AAV mice injected with AD-57553, AD-57547 andAD-58924.

FIG. 5 is a bar graph showing the relative amount of APOC3 mRNA measuredin APOC3-AAV mice previously injected with 10¹¹ genome copies of hAPOC3AAV, followed by 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg doses of AD-57553.

FIG. 6 is a bar graph showing the group averages of the relative amountof APOC3 mRNA measured in APOC3-AAV mice previously injected with 10¹¹genome copies of hAPOC3 AAV, followed by 1.25 mg/kg, 2.5 mg/kg and 5mg/kg doses of AD-57553.

FIG. 7A is a graph showing a 20-day time course of serum APOC3 proteinmeasured in APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3AAV, followed by 3 mg/kg dose of the indicated iRNAs of the invention.

FIG. 7B is a graph showing a 30-day time course of serum APOC3 proteinmeasured in APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3AAV, followed by 3 mg/kg dose of the indicated iRNAs of the invention.

FIG. 8 is a bar graph showing the amount of serum APOC3 protein measuredon day 10 in APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3AAV, followed by 3 mg/kg dose of the indicated iRNAs of the invention.

FIG. 9 is a bar graph showing the amount of serum APOC3 protein measuredon day 20 in APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3AAV, followed by 3 mg/kg dose of the indicated iRNAs of the invention.

FIG. 10 is a time course showing the amount of APOC3 protein measured inAPOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3 AAV, followedby 3 mg/kg dose of the indicated iRNAs of the invention.

FIG. 11 is a schematic showing the dosing schedule Q2W×4 used formulti-dose studies with AD-57553, AD-65696, AD-65699, AD-65703 andAD-65704.

FIG. 12A is a time course showing the amount of APOC3 protein measuredin APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3 AAV,followed by four 0.3 mg/kg doses of the indicated iRNAs of the inventionadministered according to the dosing schedule shown in FIG. 11 .

FIG. 12B is a time course showing the amount of APOC3 protein measuredin APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3 AAV,followed by four 1 mg/kg doses of the indicated iRNAs of the inventionadministered according to the dosing schedule shown in FIG. 11 .

FIG. 12C is a time course showing the amount of APOC3 protein measuredin APOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3 AAV,followed by four 3 mg/kg doses of the indicated iRNAs of the inventionadministered according to the dosing schedule shown in FIG. 11 .

FIG. 13 is a bar graph showing the relative amounts of APOC3 proteinmeasured on day 14 in APOC3-AAV mice injected with 10¹¹ genome copies ofhAPOC3 AAV, followed by single doses of 0.3 mg/kg, 1 mg/kg and 3 mg/kgof AD-65704.

FIG. 14 is a bar graph showing the relative amounts of APOC3 proteinmeasured 20 days after last dose in APOC3-AAV mice injected with 10¹¹genome copies of hAPOC3 AAV, followed by multiple doses of 0.3 mg/kg, 1mg/kg and 3 mg/kg of AD-65704, administered according to the dosingschedule shown in FIG. 11 .

FIG. 15 is a time course showing the amount of APOC3 protein measured inAPOC3-AAV mice injected with 10¹¹ genome copies of hAPOC3 AAV, followedby 1 mg/kg dose of the indicated iRNAs of the invention.

FIG. 16A is a bar graph showing the relative amounts of APOC3 proteinmeasured on day 10 in APOC3-AAV mice injected with 10¹¹ genome copies ofhAPOC3 AAV, followed by a 1 mg/kg single dose of the indicated iRNAs ofthe invention.

FIG. 16B is a bar graph showing the relative amounts of APOC3 proteinmeasured on day 24 in APOC3-AAV mice injected with 10¹¹ genome copies ofhAPOC3 AAV, followed by a 1 mg/kg single dose of the indicated iRNAs ofthe invention.

FIG. 17A is a bar graph showing the relative amounts of serum APOC3protein measured on day 14 in APOC3-AAV mice injected with 10¹¹ genomecopies of hAPOC3 AAV, followed by a single 1 mg/kg dose of the indicatediRNAs.

FIG. 17B is a graph showing the amount of serum APOC3 protein at days 0,14, 28, and 42 relative to pre-dose levels measured in APOC3-AAV miceinjected with 10¹¹ genome copies of hAPOC3 AAV, followed by a single 1mg/kg dose of the indicated iRNAs.

FIG. 18A is a graph showing the amounts, relative to pre-dose on day −7,of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, 36, 43,57, 64, and 71 in Cynomolgus monkeys following a single 1 mg/kg weeklydose of AD-65704 for 8 weeks (QW×8).

FIG. 18B is a graph showing the amounts, relative to pre-dose on day −7,of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 inCynomolgus monkeys following a single 1 mg/kg dose of AD-65704.

FIG. 18C is a graph showing the amount, relative to pre-dose on day −7,of liver APOC3 mRNA on day 64 in Cynomolgus monkeys following a single 1mg/kg weekly dose of AD-65704 for 5 weeks (q1w×5) and the amount,relative to pre-dose on day −7, of liver APOC3 mRNA on day 12 inCynomolgus monkeys following a single 1 mg/kg dose of AD-65704.

FIG. 19A is a graph showing the amounts, relative to pre-dose on day −7,of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 inCynomolgus monkeys following a single 1 mg/kg dose of the indicatediRNAs.

FIG. 19B is a bar graph showing the amounts, relative to pre-dose on day−7, of liver APOC3 mRNA measured on day 12 in Cynomolgus monkeysfollowing a single 1 mg/kg dose of the indicated iRNAs.

FIG. 20A is a graph showing the amounts, relative to pre-dose on day −7,of serum APOC3 mRNA measured on days 1, 8, 11, 15, 22, 29, 36, 43, 50,57, 64, and 71 in Cynomolgus monkeys following a single 1 mg/kg dose ofthe indicated iRNAs and subsequent administration of a singlesubcutaneous 3 mg/kg dose of the same agent on day 36.

FIG. 20B is a bar graph showing the amounts, relative to pre-dose on day−7, of liver APOC3 mRNA measured on day 12 in Cynomolgus monkeysfollowing a single 1 mg/kg dose of the indicated iRNAs on day 1 followedby a single 3 mg/kg dose of the same iRNA agent on day 36.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA agents, e.g., double-stranded iRNAagents, and compositions that reduce or inhibit the expression of anAPOC3 gene. The gene may be within a cell, e.g., a cell within asubject, such as a human.

The present invention also provides methods for treating a subjecthaving a disorder that would benefit from inhibiting or reducing theexpression of an APOC3, e.g., an apolipoprotein C3 associated disease ordisorder, such as hypertriglyceridemia, using iRNA compositions whichinhibit or reduce the expression of the APOC3 gene.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofan APOC3 gene. The use of these iRNAs enables the targeted degradationof mRNAs of the APOC3 gene in cells. Very low dosages of the iRNAs ofthe invention, in particular, can specifically and efficiently mediateRNA interference (RNAi), resulting in significant inhibition ofexpression of the APOC3 gene. Using in vitro and in vivo assays, thepresent inventors have demonstrated that iRNAs targeting the APOC3 genecan mediate RNAi, resulting in significant inhibition of expression ofAPOC3 gene and in reduced levels of APOC3 protein. The present inventorshave also demonstrated that iRNAs targeting an APOC3 gene can reduce thesymptoms associated with an apolipoprotein C3 associated disorder, e.g.,lower triglyceride levels. Thus, methods and compositions includingthese iRNAs are useful for treating a subject having an apolipoproteinC3 associated disorder, such as hypertriglyceridemia.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of APOC3 gene aswell as compositions, uses, and methods for treating subjects havingdiseases and disorders that would benefit from inhibition and/orreduction of the expression of APOC3.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, the term “APOC3” refers to the well-known gene thatencodes apolipoprotein C3, as well as to its protein product, also knownin the art as HALP2 or APOCIII.

The term “APOC3” includes human APOC3, the amino acid and completecoding sequence of which may be found in for example, GenBank AccessionNo. GI:4557322 (NM_000040.1; SEQ ID NO:1); Macaca fascicularis APOC3,the amino acid and complete coding sequence of which may be found in forexample, GenBank Accession No.

GI:544489959 (XM_05579730.1, SEQ ID NO:3); Macaca mulatta APOC3, theamino acid and complete coding sequence of which may be found in forexample, GenBank Accession No. GI:297269260 (XM_001090312.2; SEQ ID NO:5); mouse (Mus musculus) APOC3, the amino acid and complete codingsequence of which may be found in for example, GenBank Accession No.GI:577019555 (NM_023114.4, SEQ ID NO:7); rat (Rattus norvegicus) APOC3,the amino acid and complete coding sequence of which may be found in forexample, GenBank Accession No. GI:402534545 (NM_012501.2, SEQ ID NO:9);and rabbit (Oryctolagus cuniculus), GenBank Accession No. GI:655601498(XM_002708371.2, SEQ ID NO:11).

Additional examples of APOC3 mRNA sequences are readily availablethrough publicly available databases, e.g., GenBank, UniProt, OMIM, andthe Macaca genome project web site.

The term “APOC3,” as used herein, also refers to naturally occurring DNAsequence variations of the APOC3 gene, such as a single nucleotidepolymorphism (SNP) in the APOC3 gene. Exemplary SNPs in the APOC3 DNAsequence may be found through the dbSNP database available atwww.ncbi.nlm.nih.gov/projects/SNP/. Non-limiting examples of sequencevariations within the APOC3 gene include, for example, the twovariations rs2854116 and rs2854117, described in Petersen, K. F. et al.,(2010), N. Engl. J. Med. 362(12):1082-1089, the entire contents of whichare incorporated herein by reference.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an APOC3 gene, including mRNA that is a product of RNA processing ofa primary transcription product. In one embodiment, the target portionof the sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of an APOC3gene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 3). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of APOC3 in a cell, e.g., a cell within a subject, suchas a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., an APOC3target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into double-stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processesthese dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). These siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15:188). Thus, in one aspect the invention relates to a singlestranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generatedwithin a cell and which promotes the formation of a RISC complex toeffect silencing of the target gene, i.e., an APOC3 gene. Accordingly,the term “siRNA” is also used herein to refer to an RNAi as describedabove.

In another embodiment, the RNAi agent may be a single-stranded RNA thatis introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded RNAs are described in U.S. Pat. No. 8,101,348and in Lima et al., (2012) Cell 150:883-894, the entire contents of eachof which are hereby incorporated herein by reference. Any of theantisense nucleotide sequences described herein may be used as asingle-stranded siRNA as described herein or as chemically modified bythe methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” “RNAi agent”, “RNAi”, or “dsRNA”. The term“dsRNA” refers to a complex of ribonucleic acid molecules, having aduplex structure comprising two anti-parallel and substantiallycomplementary nucleic acid strands, referred to as having “sense” and“antisense” orientations with respect to a target RNA, i.e., an APOC3gene. In some embodiments of the invention, a double-stranded RNA(dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, througha post-transcriptional gene-silencing mechanism referred to herein asRNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/or amodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, eachstrand of which comprises 20-30 nucleotides that interacts with a targetRNA sequence, e.g., an APOC3 target mRNA sequence, to direct thecleavage of the target RNA. Without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into19-23 base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” RNAi agent is a dsRNA that is double-strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withnucleotide overhangs at one end (i.e., agents with one overhang and oneblunt end) or with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a APOC3 mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an APOC3 nucleotide sequence, asdefined herein. Where the region of complementarity is not fullycomplementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding APOC3). For example, a polynucleotideis complementary to at least a part of an APOC3 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding APOC3.

Accordingly, in some embodiments, the antisense polynucleotidesdisclosed herein are fully complementary to the target APOC3 sequence.In other embodiments, the sense strand polynucleotides and/or theantisense polynucleotides disclosed herein are substantiallycomplementary to the target APOC3 sequence and comprise a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs:1-12, or a fragment of any one of SEQ ID NOs:1-12,such as about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which is complementary to a target APOC3 sequence, andwherein the sense strand polynucleotide comprises a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs:1-12, or a fragment of any one of SEQ ID NOs:1-12,such as about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary. In another embodiment,an RNAi agent of the invention includes an antisense strand that issubstantially complementary to the target APOC3 sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to the equivalent region of the nucleotidesequence of any one of SEQ ID NOs:1-12, or a fragment of any one of SEQID NOs:1-12, such as about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

In one aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisense nucleicacid molecule that inhibits a target mRNA via an antisense inhibitionmechanism. The single-stranded antisense nucleic acid molecule iscomplementary to a sequence within the target mRNA. The single-strandedantisense oligonucleotides can inhibit translation in a stoichiometricmanner by base pairing to the mRNA and physically obstructing thetranslation machinery, see Dias, N. et al., (2002) Mol Cancer Ther1:347-355. The single-stranded antisense nucleic acid molecule may beabout 15 to about 30 nucleotides in length and have a sequence that iscomplementary to a target sequence. For example, the single-strandedantisense nucleic acid molecule may comprise a sequence that is at leastabout 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from anyone of the antisense sequences described herein.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in APOC3 expression; a human at risk for adisease, disorder or condition that would benefit from reduction inAPOC3 expression; a human having a disease, disorder or condition thatwould benefit from reduction in APOC3 expression; and/or human beingtreated for a disease, disorder or condition that would benefit fromreduction in APOC3 expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with unwanted orexcessive APOC3 expression, e.g., hypertriglyceridemia (or hightriglyceride levels). Such symptoms may include, e.g., skin symptoms(e.g., eruptive xanthoma); eye abnormalities (e.g., lipemia retinalis);hepatosplenomegaly (enlargement of the liver and spleen); neurologicalsymptoms; or attacks of abdominal pain that may be mild episodes ofpancreatitis. Other symptoms associated with unwanted or excessive APOC3expression may also include any symptom of a disease, disorder orcondition that may be caused by, be associated with, or be a consequenceof hypertriglyceridemia, e.g., non-alcoholic fatty liver disease,non-alcoholic steatohepatitis, polycystic ovary syndrome, kidneydisease, obesity, type 2 diabetes mellitus (insulin resistance),artherosclerosis, cardiovascular disease or pancreatitis. “Treatment”can also mean prolonging survival as compared to expected survival inthe absence of treatment.

The term “lower” in the context of the level of APOC3 in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more and is preferably down to a level accepted aswithin the range of normal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, may be treated or amelioratedby a reduction in expression of an APOC3 gene, refers to a reduction inthe likelihood that a subject will develop a symptom associated withsuch a disease, disorder, or condition, e.g., a symptom of unwanted orexcessive APOC3 expression, such as hypertriglyceridemia. The likelihoodof developing, e.g., hypertriglyceridemia, is reduced, for example, whenan individual having one or more risk factors for hypertriglyceridemiaeither fails to develop hypertriglyceridemia or developshypertriglyceridemia with less severity relative to a population havingthe same risk factors and not receiving treatment as described herein.The failure to develop a disease, disorder or condition, or thereduction in the development of a symptom associated with such adisease, disorder or condition (e.g., by at least about 10% on aclinically accepted scale for that disease or disorder), or theexhibition of delayed symptoms delayed (e.g., by days, weeks, months oryears) is considered effective prevention.

As used herein, the term “apolipoprotein C3-associated disease” or“APOC3-associated disease,” is a disease, disorder or a condition thatis caused by, or is associated with, unwanted or excessive APOC3expression. The term “APOC3-associated disease” includes a disease,disorder or condition that may be treated or ameliorated by a reductionin APOC3 expression. The term APOC3-associated disease” includeshypertriglyceridemia, or a high triglyceride levels.

The levels of triglycerides in a serum of a subject, e.g., a humansubject, that may be indicative of hypertriglyceridemia are described inOh, R. C. et al., (2007) American Family Physician, 75(9):1366-1371.Specifically, hypertriglyceridemia may be associated with“borderline-high serum triglyceride levels” (i.e., 150 to 199 mg per dLor 1.70 to 2.25 mmol per L); “high serum triglyceride levels” (i.e., 200to 499 mg per dL or 2.26 to 5.64 mmol per L); or “very high triglyceridelevels” (i.e., 500 mg per dL or higher (or 5.65 mmol per L or higher)

In one embodiment, an APOC3-associated disease is primaryhypertriglyceridemia. “Primary triglyceridemia” results fromenvironmental or genetic causes (e.g., a result of no obvious underlyingmedical cause). Exemplary diseases characterized as primaryhypertriglyceridemias include, but are not limited to, familialchylomicronemia (hyperlipoproteinemia type I), primary mixedhyperlipidemia (type 5), familial hypertriglyceridemia(hyperlipoproteinemia type 4), familial combined hyperlipoproteinemia(type 2B) and familial dysbetalipoproteinemia (hyperlipoproteinemia type3).

In another embodiment, an APOC3-associated disease is secondaryhypertriglyceridemia. “Secondary triglyceridemia” is caused by, or beassociated with, other underlying disorders and conditions. Suchdisorders and/or conditions include, e.g., obesity, metabolic syndrome,diabetes, fatty liver, alcohol use, renal disease, pregnancy,nonalcoholic fatty liver disorder, hypothyroidism, paraproteinemias(such as hypergammaglobulinemia in macroglobulinemia, myeloma, lymphomaand lymphocytic leukemias), autoimmune disorders (such as systemic lupuserythematosis), intake of medications (such as antiretroviral drugs,including ritonavir and lopinavir, and antipsychotic medications,including clozapine and olanzapine), see G. Yuan et al., (2007) CanadianMedical Association Journal, 176(8):1113-1120.

Any disorder that may be a cause of hypertriglyceridemia (e.g.,secondary hypertriglyceridemia) or that may be a consequence ofhypertriglyceridemia (e.g., primary or secondary hypertriglyceridemia)is encompassed by the term “APOC3-associated disease”. Non-limitingexamples of APOC3-associated diseases include metabolic disorders, e.g.,non-alcoholic fatty liver disease, non-alcoholic steatohepatitis,polycystic ovary syndrome, kidney disease, obesity, type 2 diabetesmellitus (insulin resistance); hypertension; cardiovascular disorders,e.g., artherosclerosis; and pancreatitis, e.g., acute pancreatitis.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of anAPOC3 gene. In one embodiment, the iRNA agent includes double-strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anAPOC3 gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human having an APOC3-associated disease, e.g.,hypertriglyceridemia. The dsRNA includes an antisense strand having aregion of complementarity which is complementary to at least a part ofan mRNA formed in the expression of an APOC3 gene. The region ofcomplementarity is about 30 nucleotides or less in length (e.g., about30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides orless in length). Upon contact with a cell expressing the APOC3 gene, theiRNA inhibits the expression of the APOC3 gene (e.g., a human, aprimate, a non-primate, or a bird APOC3 gene) by at least about 10% asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an APOC3gene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25,20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengthsintermediate to the above recited ranges and lengths are alsocontemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 20nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well-known in the art that dsRNAslonger than about 21-23 nucleotides in length may serve as substratesfor Dicer. As the ordinarily skilled person will also recognize, theregion of an RNA targeted for cleavage will most often be part of alarger RNA molecule, often an mRNA molecule. Where relevant, a “part” ofan mRNA target is a contiguous sequence of an mRNA target of sufficientlength to allow it to be a substrate for RNAi-directed cleavage (i.e.,cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target APOC3 expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandand the corresponding antisense strand are each selected from the groupof sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A,11B, 12, and 13. In this aspect, one of the two sequences iscomplementary to the other of the two sequences, with one of thesequences being substantially complementary to a sequence of an mRNAgenerated in the expression of an APOC3 gene. As such, in this aspect, adsRNA will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand in any one of Tables 4A, 4B, 5, 8, 9, 10,11A, 11B, 12, and 13, and the second oligonucleotide is described as thecorresponding antisense strand of the sense strand in any one of Tables4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. In one embodiment, thesubstantially complementary sequences of the dsRNA are contained onseparate oligonucleotides. In another embodiment, the substantiallycomplementary sequences of the dsRNA are contained on a singleoligonucleotide.

It will be understood that, although some of the sequences in Tables 4A,4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, are described as modified and/orconjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNAof the invention, may comprise any one of the sequences set forth inTables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 that is un-modified,un-conjugated, and/or modified and/or conjugated differently thandescribed therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 4A, 4B, 5, 8, 9,10, 11A, 11B, 12, and 13, dsRNAs described herein can include at leastone strand of a length of minimally 21 nucleotides. It can be reasonablyexpected that shorter duplexes having one of the sequences of any one ofTables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 minus only a fewnucleotides on one or both ends can be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a sequence of atleast 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derivedfrom one of the sequences of any one of Tables 4A, 4B, 5, 8, 9, 10, 11A,11B, 12, and 13, and differing in their ability to inhibit theexpression of a APOC3 gene by not more than about 5, 10, 15, 20, 25, or30% inhibition from a dsRNA comprising the full sequence, arecontemplated to be within the scope of the present invention.

In addition, the RNAs provided in any one of Tables 4A, 4B, 5, 8, 9, 10,11A, 11B, 12, and 13 identify a site(s) in a APOC3 transcript that issusceptible to RISC-mediated cleavage. As such, the present inventionfurther features iRNAs that target within one of these sites. As usedherein, an iRNA is said to target within a particular site of an RNAtranscript if the iRNA promotes cleavage of the transcript anywherewithin that particular site. Such an iRNA will generally include atleast about 15 contiguous nucleotides from one of the sequences providedin any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 111B, 12, and 13 coupledto additional nucleotide sequences taken from the region contiguous tothe selected sequence in a APOC3 gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 4A,4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 represent effective targetsequences, it is contemplated that further optimization of inhibitionefficiency can be achieved by progressively “walking the window” onenucleotide upstream or downstream of the given sequences to identifysequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, furtheroptimization could be achieved by systematically either adding orremoving nucleotides to generate longer or shorter sequences and testingthose sequences generated by walking a window of the longer or shortersize up or down the target RNA from that point. Again, coupling thisapproach to generating new candidate targets with testing foreffectiveness of iRNAs based on those target sequences in an inhibitionassay as known in the art and/or as described herein can lead to furtherimprovements in the efficiency of inhibition. Further still, suchoptimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of an APOC3 gene, generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an APOC3 gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of an APOC3gene is important, especially if the particular region ofcomplementarity in an APOC3 gene is known to have polymorphic sequencevariation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is unmodified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified nucleotides. iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

In some aspects of the invention, substantially all of the nucleotidesof an iRNA of the invention are modified and the iRNA agents comprise nomore than 10 nucleotides comprising 2′-fluoro modifications (e.g., nomore than 9 2′-fluoro modifications, no more than 8 2′-fluoromodifications, no more than 7 2′-fluoro modifications, no more than 62′-fluoro modifications, no more than 5 2′-fluoro modifications, no morethan 4 2′-fluoro modifications, no more than 5 2′-fluoro modifications,no more than 4 2′-fluoro modifications, no more than 3 2′-fluoromodifications, or no more than 2 2′-fluoro modifications). For example,in some embodiments, the sense strand comprises no more than 4nucleotides comprising 2′-fluoro modifications (e.g., no more than 32′-fluoro modifications, or no more than 2 2′-fluoro modifications). Inother embodiments, the antisense strand comprises no more than 6nucleotides comprising 2′-fluoro modifications (e.g., no more than 52′-fluoro modifications, no more than 4 2′-fluoro modifications, no morethan 4 2′-fluoro modifications, or no more than 2 2′-fluoromodifications). In other aspects of the invention, all of thenucleotides of an iRNA of the invention are modified and the iRNA agentscomprise no more than 10 nucleotides comprising 2′-fluoro modifications(e.g., no more than 9 2′-fluoro modifications, no more than 8 2′-fluoromodifications, no more than 7 2′-fluoro modifications, no more than 62′-fluoro modifications, no more than 5 2′-fluoro modifications, no morethan 4 2′-fluoro modifications, no more than 5 2′-fluoro modifications,no more than 4 2′-fluoro modifications, no more than 3 2′-fluoromodifications, or no more than 2 2′-fluoro modifications).

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(m)O]_(m)CH₃, O(CH₂)_(-n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2 N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2 C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. patents and US patenttent Publicationsthat teach the preparation of locked nucleic acid nucleotides include,but are not limited to, the following: U.S. Pat. Nos. 6,268,490;6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207;7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457;8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618;and US 2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and 3-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in WO 2013/075035, filed on Nov. 16, 2012, the entirecontents of which are incorporated herein by reference. As shown hereinand in PCT Publication No. WO 2013/075035, a superior result may beobtained by introducing one or more motifs of three identicalmodifications on three consecutive nucleotides into a sense strandand/or antisense strand of an RNAi agent, particularly at or near thecleavage site. In some embodiments, the sense strand and antisensestrand of the RNAi agent may otherwise be completely modified. Theintroduction of these motifs interrupts the modification pattern, ifpresent, of the sense and/or antisense strand. The RNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand. The resulting RNAi agents present superior genesilencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an RNAi agent, the gene silencingactivity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double-stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., apolipoprotein C3(APOC3) gene) in vivo. The RNAi agent comprises a sense strand and anantisense strand. Each strand of the RNAi agent may range from 12-30nucleotides in length. For example, each strand may be between 14-30nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides inlength, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides inlength, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In one embodiment, everynucleotide in the sense strand and the antisense strand of the RNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In one embodiment each residue is independentlymodified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif.Optionally, the RNAi agent further comprises a ligand (preferablyGalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3′terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3′ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region which is at least 25 nucleotides in length, and thesecond strand is sufficiently complementary to a target mRNA along atleast 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing acitivty to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mistmatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):

5′n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′  (Ib);

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q)3′  (Ic); or

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides. Each of X, Y and Z may be the same or different from eachother.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′n_(p)-N_(a)—YYY—N_(a)-n_(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n_(q′)-N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′n_(q′)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(a)′-n_(p)′3′  (IIb);

5′n_(q′)-N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n_(p)′3′  (IIc); or

5′n_(q′)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n_(p)′3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may berepresented by the formula:

5′n_(p)′—N_(a)—Y′Y′Y′—N_(a)′-n_(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(i)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′n_(p)-N_(a)—YYY—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—Y′Y′Y′—N_(a)′n_(q′)5′   (IIIa)

5′n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)′n_(q′)5′   (IIIb)

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n_(q′)5′   (IIIc)

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)-n_(q)5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′ >0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

In certain embodiments, an RNAi agent of the invention may contain a lownumber of nucleotides containing a 2′-fluoro modification, e.g., 10 orfewer nucleotides with 2′-fluoro modification. For example, the RNAiagent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with a2′-fluoro modification. In a specific embodiment, the RNAi agent of theinvention contains 10 nucleotides with a 2′-fluoro modification, e.g., 4nucleotides with a 2′-fluoro modification in the sense strand and 6nucleotides with a 2′-fluoro modification in the antisense strand. Inanother specific embodiment, the RNAi agent of the invention contains 6nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a2′-fluoro modification in the sense strand and 2 nucleotides with a2′-fluoro modification in the antisense strand.

In other embodiments, an RNAi agent of the invention may contain anultra low number of nucleotides containing a 2′-fluoro modification,e.g., 2 or fewer nucleotides containing a 2′-fluoro modification. Forexample, the RNAi agent may contain 2, 1 of 0 nucleotides with a2′-fluoro modification. In a specific embodiment, the RNAi agent maycontain 2 nucleotides with a 2′-fluoro modification, e.g., 0 nucleotideswith a 2-fluoro modification in the sense strand and 2 nucleotides witha 2′-fluoro modification in the antisense strand.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. Theseagents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennapedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 24). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 25) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 26) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 27)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include AGTand above (e.g., AGT, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., AGT, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the presentinvention include those described in PCT Publication Nos. WO 2014/179620and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having an APOC3-associated disease) can be achieved in anumber of different ways. For example, delivery may be performed bycontacting a cell with an iRNA of the invention either in vitro or invivo. In vivo delivery may also be performed directly by administering acomposition comprising an iRNA, e.g., a dsRNA, to a subject.Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the APOC3 gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an APOC3 gene. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery,e.g., by subcutaneous (SC) or intravenous (IV) delivery. Another exampleis compositions that are formulated for direct delivery into the brainparenchyma, e.g., by infusion into the brain, such as by continuous pumpinfusion. The pharmaceutical compositions of the invention may beadministered in dosages sufficient to inhibit expression of an APOC3gene. In general, a suitable dose of an iRNA of the invention will be inthe range of about 0.001 to about 200.0 milligrams per kilogram bodyweight of the recipient per day, generally in the range of about 1 to 50mg per kilogram body weight per day. For example, the dsRNA can beadministered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg,about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg,about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about50 mg/kg per single dose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the dsRNA is administered at a dose of about 0.1to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the dsRNA isadministered at a dose of about 10 mg/kg to about 30 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered, e.g., subcutaneously orintravenously, a single therapeutic amount of iRNA, such as about 0.1,0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375,0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65,0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925,0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29,29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In some embodiments, subjects are administered, e.g., subcutaneously orintravenously, multiple doses of a therapeutic amount of iRNA, such as adose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A multi-dose regimine mayinclude administration of a therapeutic amount of iRNA daily, such asfor two days, three days, four days, five days, six days, seven days, orlonger.

In other embodiments, subjects are administered, e.g., subcutaneously orintravenously, a repeat dose of a therapeutic amount of iRNA, such as adose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. A repeat-dose regiminemay include administration of a therapeutic amount of iRNA on a regularbasis, such as every other day, every third day, every fourth day, twicea week, once a week, every other week, or once a month.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate iRNA. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natd. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNA agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA agentcan be delivered, for example, subcutaneously by infection in order todeliver iRNA agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C22 alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 Exemplary lipid-dsRNA formulations. cationic lipid/non-cationiclipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic LipidLipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA)(57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA-6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:1l-yl)ethylazanediyl)didodecan-2-ol (TechG1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and antioxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise, a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically, microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al., Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),iRNAMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-iRNA mechanism and which are useful intreating an APOC3-associated disorder. Examples of such agents include,but are not limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby APOC3 expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VII. Methods of the Invention

The present invention provides therapeutic and prophylactic methodswhich include administering to a subject having, or prone to developing,an APOC3-associated disease, disorder, and/or condition (e.g.,hypertriglyceridemia), pharmaceutical compositions comprising an iRNAagent, or vector comprising an iRNA of the invention.

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in APOC3expression, e.g., hypertriglyceridemia and other APOC-3 associateddiseases, e.g., non-alcoholic fatty liver disease, non-alcoholicsteatohepatitis, polycystic ovary syndrome, kidney disease, obesity,type 2 diabetes mellitus (insulin resistance); hypertension;cardiovascular disorders, e.g., artherosclerosis; and pancreatitis,e.g., acute pancreatitis

The treatment methods (and uses) of the invention include administeringto the subject, e.g., a human, a therapeutically effective amount of aniRNA agent targeting an APOC3 gene or a pharmaceutical compositioncomprising an iRNA agent targeting an APOC3 gene, thereby treating thesubject having a disorder that would benefit from reduction in APOC3expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin APOC3 expression, e.g., a APOC3-associated disease, such ashypertriglyceridemia and other diseases that may be caused by, beassociated with, or be a consequence of hypertriglyceridemia. The latterdiseases include, but are not limited to, non-alcoholic fatty liverdisease, non-alcoholic steatohepatitis, polycystic ovary syndrome,kidney disease, obesity, type 2 diabetes mellitus (insulin resistance),artherosclerosis, cardiovascular disease or pancreatitis. The methodsinclude administering to the subject a therapeutically effective amountof the iRNA agent, e.g., dsRNA, or vector of the invention, therebypreventing at least one symptom in the subject having a disorder thatwould benefit from reduction in APOC3 expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of an iRNA agent of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of APOC3 expression.

In a further aspect, the present invention provides uses of an iRNAagent, e.g., a dsRNA, of the invention targeting an APOC3 gene orpharmaceutical composition comprising an iRNA agent targeting an APOC3gene in the manufacture of a medicament for treating a subject, e.g., asubject that would benefit from a reduction and/or inhibition of APOC3expression, such as a subject having a disorder that would benefit fromreduction in APOC3 expression, e.g., a APOC3-associated disease, such ashypertriglyceridemia and other diseases that may be caused by, beassociated with, or be a consequence of hypertriglyceridemia. The latterdiseases may include, but are not limited to, non-alcoholic fatty liverdisease, non-alcoholic steatohepatitis, polycystic ovary syndrome,kidney disease, obesity, type 2 diabetes mellitus (insulin resistance),artherosclerosis, cardiovascular disease or pancreatitis.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of APOC3 expression, such as a APOC3-associated disease,e.g., hypertriglyceridemia and other diseases that may be caused by, beassociated with, or be a consequence of hypertriglyceridemia. The latterdiseases may include, but are not limited to, non-alcoholic fatty liverdisease, non-alcoholic steatohepatitis, polycystic ovary syndrome,kidney disease, obesity, type 2 diabetes mellitus (insulin resistance),artherosclerosis, cardiovascular disease or pancreatitis.

In a further aspect, the present invention provides uses of an iRNAagent of the invention in the manufacture of a medicament for preventingat least one symptom in a subject suffering from a disorder that wouldbenefit from a reduction and/or inhibition of APOC3 expression, such asan APOC3-associated disease, e.g., hypertriglyceridemia and otherdiseases that may be caused by, be associated with, or be a consequenceof hypertriglyceridemia. The latter diseases may include, but are notlimited to, non-alcoholic fatty liver disease, non-alcoholicsteatohepatitis, polycystic ovary syndrome, kidney disease, obesity,type 2 diabetes mellitus (insulin resistance), artherosclerosis,cardiovascular disease or pancreatitis.

In one embodiment, an iRNA agent targeting APOC3 is administered to asubject having a APOC3-associated disease such that APOC3 levels, e.g.,in a cell, tissue, blood or other tissue or fluid of the subject arereduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%or more when the dsRNA agent is administered to the subject.

The methods and uses of the invention include administering acomposition described herein such that expression of the target APOC3gene is decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18,24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80hours. In one embodiment, expression of the target APOC3 gene isdecreased for an extended duration, e.g., at least about two, three,four, five, six, seven days or more, e.g., about one week, two weeks,three weeks, or about four weeks or longer.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with anAPOC3-associated disease. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of hypertriglyceridemia may be assessed,for example, by periodic monitoring of blood triglyceride levels.Comparison of the later readings with the initial readings provide aphysician an indication of whether the treatment is effective. It iswell within the ability of one skilled in the art to monitor efficacy oftreatment or prevention by measuring any one of such parameters, or anycombination of parameters. In connection with the administration of aniRNA targeting APOC3 or pharmaceutical composition thereof, “effectiveagainst” an APOC3-associated disease indicates that administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such asimprovement of symptoms, a cure, a reduction in disease, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating aAPOC3-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed. Suitable animal models ofan APOC3-associated disease include any animal models that have, e.g.,hypertriglyceridemia. Such animal models include, e.g., transgenic miceexpressing the human apolipoprotein C2 (APOC2) gene (such as mice of thestrain B6; CBA-Tg(APOC2)2Bres/J or B6.Cg-Tg(APOC2)2Bres/J available fromthe Jackson Laboratory in Bar Harbor, Me.); transgenic mice expressingthe human apoprotein C3 (APOC3) gene (such as mice of the strain B6;CBA-Tg(APOC3)3707Bres/J, available from the Jackson Laboratory);homozygous fatty liver dystrophy (fld) mice (such as mice of the strainC3H/HeJ-Lpin1^(fld-2J)/J, available from the Jackson Laboratory); micehomozygous for the ENU-induced missense mutation called Sec61a1^(Y344H)(such as mice of the strain C57BL/6J-Sec61a1^(m1Gek)/J, available fromthe Jackson Laboratory); diet-induced obese mice (such as mice of thestrain C57BL/6J fed 60% kcal fat diet, available from the JacksonLaboratory or mice of the strain C57BL/6NTac fed 60% kcal fat diet,available from Taconic); and transgenic C57Bl/6 mice overexpressing thehuman ApoC3 gene under liver-specific TBG promotor or transgenic C57Bl/6mice as described herein.

In certain embodiments, an experimental animal model suitable fortesting the efficacy of an iRNA or a formulation comprising an iRNA ofthe invention includes a rabbit. Exemplary rabbit models include, e.g.,a Watanabe heritable hyperlipidemic (WHHL) rabbit. The WHHL rabbit is ananimal model for hypercholesterolemia due to a deficiency of low-densitylipoprotein (LDL) receptors. The characteristics of the WHHL rabbit andthe history of studies conducted using the WHHL rabbits are described,e.g., by Shiomi, M. and Ito, T., Artherosclerosis (2009), 207(1):1-7,the entire contents of which are hereby incorporated herein byreference. WHHL rabbits exhibit increased cholesterol and triglyceridelevels as shown below;

Plasma Lipid (mg/dL) Normal Rabbits WHHL Rabbits Total cholesterol 41 ±10 810 ± 110 Triglyceride 34 ± 19 417 ± 117

In certain embodiments, a WHHL rabbit may be a preferred animal modelfor studying inhibition of APOC3 expression because the WHHL rabbitexhibits a more human-like lipid profile than other animal models andmay contribute to the understanding of the relationship between ApoC3knock-down and lowering of triglycerides and may inform dosing for thestudies involving non-human primates. The comparison of enzyme andlipoprotein profiles among various animal species is presented in Table2 below.

TABLE 2 Differences in Enzymes and Lipoprotein Profiles Among AnimalSpecies WHHL Enzyme or Lipoprotein Mice and Rats Human Rabbit Mainplasma lipoproteins HDL or VLDL LDL LDL ApoB on VLDL ApoB-48 & ApoB-100ApoB-100 apoB-100 ApoB Editing enzyme Ileum and liver Ileum ileum CETPNone Yes Yes Hepatic lipase In plasma Binding Binding to cell to cellmembrane membrane Atherosclerosis Resistance Susceptible SpontaneousLipid-rich/ Various Various Collagen-poor lesions lesionsHypocholesterolemic No effect or Effective Effective effects of statinselevation

Other exemplary rabbit models that may be suitable for testing theefficacy of an iRNA or a formulation comprising an iRNA of the inventioninclude, e.g., a diet induced obese rabbit. Diet induced obese rabbitshave been previously described in the literature by, e.g., Taylor andFan, Front. Biosci. (1997), 2:298-308; Carroll et al., Am. J. Physiol.(1996), 271:H373-8; Antic et al., Am. J. Hypertens. (2000), 13:556-9;Carroll et al., Acta Physiol. Scand. (2004), 181:183-91; and Rong etal., Arterioscler. Thromb. Vasc. Biol. (1999), 19:2179-88, the entirecontents of which are incorporated herein by reference.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg,1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kgdsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kgdsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kgdsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kgdsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kgdsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kgdsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kgdsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kgdsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kgdsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kgdsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kgdsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kgdsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kgdsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kgdsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kgdsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kgdsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kgdsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kgdsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kgdsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA,30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about50 mg/kg dsRNA. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes a modification (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides), including one such motif at or near the cleavage site ofthe agent, six phosphorothioate linkages, and a ligand, such an agent isadministered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 toabout 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or about a 25 minute period. The administrationmay be repeated, for example, on a regular basis, such as weekly,biweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration weekly or biweekly for three months, administrationcan be repeated once per month, for six months or a year or longer.

Administration of the iRNA can reduce APOC3 levels, e.g., in a cell,tissue, blood, urine or other compartment of the patient by at leastabout 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion, and monitored foradverse effects, such as an allergic reaction. In another example, thepatient can be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on APOC3 expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

An iRNA of the invention may be administered in “naked” form, where themodified or unmodified iRNA agent is directly suspended in aqueous orsuitable buffer solvent, as a “free iRNA.” A free iRNA is administeredin the absence of a pharmaceutical composition. The free iRNA may be ina suitable buffer solution. The buffer solution may comprise acetate,citrate, prolamine, carbonate, or phosphate, or any combination thereof.In one embodiment, the buffer solution is phosphate buffered saline(PBS). The pH and osmolarity of the buffer solution containing the iRNAcan be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of APOC3gene expression are those having a APOC3-associated disease or disorderas described herein.

Treatment of a subject that would benefit from a reduction and/orinhibition of APOC3 gene expression includes therapeutic andprophylactic treatment.

The invention further provides methods and uses of an iRNA agent or apharmaceutical composition thereof for treating a subject that wouldbenefit from reduction and/or inhibition of APOC3 expression, e.g., asubject having a APOC3-associated disease, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these diseases. Forexample, in certain embodiments, an iRNA targeting APOC3 is administeredin combination with, e.g., an additional agent useful in treating anAPOC3-associated disease.

Examples of additional therapeutic agents include those known to treathypertriglyceridemia and other diseases that are caused by, associatedwith or are a consequence of hypertriglyceridemia. For example, an iRNAfeatured in the invention can be administered with, e.g., an HMG-CoAreductase inhibitor (e.g., a statin), a fibrate, a bile acidsequestrant, niacin, an antiplatelet agent, an angiotensin convertingenzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartanpotassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterolacetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor,a cholesterol ester transfer protein (CETP) inhibitor, a microsomaltriglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator,a bile acid modulator, a peroxisome proliferation activated receptor(PPAR) agonist, a gene-based therapy, a composite vascular protectant(e.g., AGI-1067, from Atherogenics), a glycoprotein IIb/IIIa inhibitor,aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921,from Shionogi), a squalene synthase inhibitor, a monocytechemoattractant protein (MCP)-I inhibitor, or fish oil. ExemplaryHMG-CoA reductase inhibitors include atorvastatin (Pfizer'sLipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-MyersSquibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck'sZocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas),lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa;Schwarz Pharma's Liposcler), fluvastatin (Novartis'Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin(Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca'sCrestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical,Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g.,bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol),clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier'sLipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics),gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate(Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrantsinclude, e.g., cholestyramine (Bristol-Myers Squibb's Questran® andQuestran Light™), colestipol (e.g., Pharmacia's Colestid), andcolesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapiesinclude, e.g., immediate release formulations, such as Aventis' Nicobid,Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit.Niacin extended release formulations include, e.g., Kos Pharmaceuticals'Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agentsinclude, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel(Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine(e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Otheraspirin-like compounds useful in combination with a dsRNA targetingAPOC3 include, e.g., Asacard (slow-release aspirin, by Pharmacia) andPamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplaryangiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g.,Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplaryacyl CoA cholesterol acetyltransferase (AC AT) inhibitors include, e.g.,avasimibe (Pfizer), eflucimibe (BioMsrieux Pierre Fabre/Eli Lilly),CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterolabsorption inhibitors include, e.g., ezetimibe (Merck/Schering-PloughPharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETPinhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer),JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics).Exemplary microsomal triglyceride transfer protein (MTTP) inhibitorsinclude, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086(Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886(Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433(Wyeth-Ayerst).

Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu),Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921(Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplaryperoxisome proliferation activated receptor (PPAR) agonists include,e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555)(Mitsubishi/Johnson & Johnson), GW-409544 (LigandPharniaceuticals/GlaxoSmithKline), GW-501516 (LigandPharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and EliLilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674(Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin).Exemplary gene-based therapies include, e.g; AdGWEGF 121.10 (GenVec),ApoA1 (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics),and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte,Aventis, Xenon). Exemplary Glycoprotein IIb/IIIa inhibitors include,e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban(Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).Exemplary squalene synthase inhibitors include, e.g., BMS-1884941(Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer),CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitoris, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agentBO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivativeNyclin (Yamanouchi Pharmacuticals) are also appropriate foradministering in combination with a dsRNA featured in the invention.Exemplary combination therapies suitable for administration with a dsRNAtargeting APOC3 include, e.g., advicor (Niacin/lovastatin from KosPharmaceuticals), amlodipine/atorvastatin (Pfizer), andezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treatinghypertriglyceridemia, and suitable for administration in combinationwith a dsRNA targeting APOC3 include, e.g., lovastatin, niacin Altoprev®Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets(Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets(AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis),fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodiumLipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules(Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets(Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott),fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-PloughPharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo),colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia®Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor®Tablets (Merck).

In one embodiment, an iRNA agent is administered in combination with anezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-PloughPharmaceuticals)). In one embodiment, the iRNA agent is administered tothe patient, and then the additional therapeutic agent is administeredto the patient (or vice versa). In another embodiment, the iRNA agentand the additional therapeutic agent are administered at the same time.

The iRNA agent and an additional therapeutic agent and/or treatment maybe administered at the same time and/or in the same combination, e.g.,parenterally, or the additional therapeutic agent can be administered aspart of a separate composition or at separate times and/or by anothermethod known in the art or described herein.

The present invention also provides methods of using an iRNA agent ofthe invention and/or a composition containing an iRNA agent of theinvention to reduce and/or inhibit APOC3 expression in a cell. In otheraspects, the present invention provides an iRNA of the invention and/ora composition comprising an iRNA of the invention for use in reducingand/or inhibiting APOC3 expression in a cell. In yet other aspects, useof an iRNA of the invention and/or a composition comprising an iRNA ofthe invention for the manufacture of a medicament for reducing and/orinhibiting APOC3 expression in a cell are provided.

The methods and uses include contacting the cell with an iRNA, e.g., adsRNA, of the invention and maintaining the cell for a time sufficientto obtain degradation of the mRNA transcript of an APOC3 gene, therebyinhibiting expression of the APOC3 gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of APOC3 may bedetermined by determining the mRNA expression level of APOC3 usingmethods routine to one of ordinary skill in the art, e.g., Northernblotting, qRT-PCR, by determining the protein level of APOC3 usingmethods routine to one of ordinary skill in the art, such as Westernblotting, immunological techniques, flow cytometry methods, ELISA,and/or by determining a biological activity of APOC3.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an APOC3 gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

APOC3 expression may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the APOC3 gene of the mammal to be treated. When theorganism to be treated is a human, the composition can be administeredby any means known in the art including, but not limited tosubcutaneous, intravenous, oral, intraperitoneal, or parenteral routes,including intracranial (e.g., intraventricular, intraparenchymal andintrathecal), intramuscular, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by subcutaneousor intravenous infusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof APOC3, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an APOC3 gene in a mammal, e.g., a human.The present invention also provides a composition comprising an iRNA,e.g., a dsRNA, that targets an APOC3 gene in a cell of a mammal for usein inhibiting expression of the APOC3 gene in the mammal. In anotheraspect, the present invention provides use of an iRNA, e.g., a dsRNA,that targets an APOC3 gene in a cell of a mammal in the manufacture of amedicament for inhibiting expression of the APOC3 gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an iRNA, e.g., a dsRNA, that targets an APOC3gene in a cell of the mammal and maintaining the mammal for a timesufficient to obtain degradation of the mRNA transcript of the APOC3gene, thereby inhibiting expression of the APOC3 gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sampleof the iRNA-administered subject by any methods known it the art, e.g.qRT-PCR, described herein. Reduction in protein production can beassessed by any methods known it the art and by methods, e.g., ELISA orWestern blotting, described herein. In one embodiment, a puncture liverbiopsy sample serves as the tissue material for monitoring the reductionin APOC3 gene and/or protein expression. In another embodiment, a bloodsample serves as the tissue material for monitoring the reduction inAPOC3 gene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target invivo following administration of iRNA agent is done by performing5′-RACE or modifications of the protocol as known in the art (Lasham Aet al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al.(2006) Nature 441: 111-4).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and the Sequence Listing, arehereby incorporated herein by reference.

EXAMPLES Materials and Methods

The following materials and methods were used in the Examples.

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

APOC3 siRNA sequences were synthesized at 1 μmol scale on Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500 Å) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F, 2′-O-Methyl, GNA (glycol nucleic acids),5′phosphate and abasic modifications were introduced employing thecorresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated singlestrands was performed on a GalNAc modified CPG support. Custom CPGuniversal solid support was used for the synthesis of antisense singlestrands. Coupling time for all phosphoramidites (100 mM in acetonitrile)was 5 min employing 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 Min acetonitrile). Phosphorothioate linkages were generated using a 50 mMsolution of 3-((Dimethylamino-methylidene) amino)-3H-1, 2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass.,USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3minutes. All sequences were synthesized with final removal of the DMTgroup (“DMT off”).

APOC3 sequences for in vitro screening assays were initially synthesizedusing ‘TOFFEE-6PS’ motif. In TOFFEE-6PS design, sense strands are madeof 21 nucleotides in length, with GalNAc ligand at the 3′end, twophosporothioates at the 5′end and a triplet of 2′F nucleotides atpositions 9, 10 and 11. The antisense sequences in TOFFEE-6PS design are23 nucleotides in length; contain a 3 nucleotide triplet of 2′-OMenucleotides at positions 11, 12 and 13 with two phosphorothioates at 3′and 5′ ends respectively.

Upon completion of the solid phase synthesis, oligoribonucleotides werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagent at 60° C. for 20minutes. For sequences containing 2′ ribo residues (2′-OH) that areprotected with tert-butyl dimethyl silyl (TBDMS) group, a second stepdeprotection was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To the methylamine deprotection solution, 200 μL ofdimethyl sulfoxide (DMSO) and 300 μl TEA.3HF reagent was added, and thesolution was incubated for additional 20 minutes at 60° C. At the end ofcleavage and deprotection step, the synthesis plate was allowed to cometo room temperature and was precipitated by addition of 1 mL ofacetontile:ethanol mixture (9:1). The plates were cooled at −80° C. for2 hrs, and the superanatant was decanted carefully with the aid of amulti channel pipette. The oligonucleotide pellet was re-suspended in 20mM NaOAc buffer and were desalted using a 5 mL HiTrap size exclusioncolumn (GE Healthcare) on an AKTA Purifier System equipped with an A905autosampler and a Frac 950 fraction collector. Desalted samples werecollected in 96 well plates. Samples from each sequence were analyzed byLC-MS to confirm the identity, UV (260 nm) for quantification and aselected set of samples by IEX chromatography to determine purity.

Annealing of APOC3 single strands was performed on a Tecan liquidhandling robot. Equimolar mixture of sense and antisense single strandswere combined and annealed in 96 well plates. After combining thecomplementary single strands, the 96 well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 10 μM in 1×PBS and then submitted for in vitroscreening assays.

Cell Culture and 96-Well Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ Hep3Bcells were then added to the siRNA mixture. Cells were incubated foreither 24 or 120 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and response experiments were done over a range of dosesfrom 10 nM to 36 fM final duplex concentration over 8, 6-fold dilutions.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl ApoC3 TaqMan probe(Applied Biosystems cat #Hs00163644_m1) and 5 μl Lightcycler 480 probemaster mix (Roche Cat #04887301001) per well in a 384 well plates (Rochecat #04887301001). Real time PCR was done in a LightCycler480 Real TimePCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested intwo independent transfections and each transfection was assayed induplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells.

The sense and antisense sequences ofAD-1955 are:

SENSE: (SEQ ID NO: 28) cuuAcGcuGAGuAcuucGAdTsdT ANTISENSE:(SEQ ID NO: 29) UCGAAGuACUcAGCGuAAGdTsdT

TABLE 3 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that, unless otherwiseindicated, these monomers, when present in an oligonucleotide, aremutually linked by 5′-3′-phosphodiester bonds. AbbreviationNucleotide(s) A Adenosine-3′-phosphate Af2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]- 4-hydroxyprolinolHyp-(GalNAc-alkyl)3 dT 2′-deoxythymidine-3′-phosphate VP Vinyl phosphateAgn Adenosine-glycol nucleic acid (GNA) Tgn Thymidine-glycol nucleicacid (GNA) S-Isomer Y44 2-hydroxymethyl-tetrahydrofurane-5-phosphate dG2′-deoxyguanosine-3′-phosphate

Example 1. iRNA Synthesis

Generation of the Initial Set of iRNA Agents Targeting APOC3

A set of iRNAs targeting the human APOC3, “apolipoprotein C-III” (human:NCBI refseqID NM_000040.1), as well as tox-species APOC3 orthologs(cynomolgus monkey: XM_005579730; rhesus monkey, XM_001090312.2; mouse:NM_023114; rat, NM_012501) were designed using custom R and Pythonscripts. The human APOC3 REFSEQ mRNA has a length of 533 bases. Therationale and method for the set of siRNA designs is as follows: thepredicted efficacy for every potential 19mer iRNA from position 47through position 533 was determined with a linear model derived thedirect measure of mRNA knockdown from more than 20,000 distinct iRNAdesigns targeting a large number of vertebrate genes. Subsets of theAPOC3 siRNAs were designed with perfect or near-perfect matches betweenhuman, cynomolgus and rhesus monkey. A further subset was designed withperfect or near-perfect matches to mouse and rat APOC3 orthologs. Foreach strand of the iRNA, a custom Python script was used in a bruteforce search to measure the number and positions of mismatches betweenthe siRNA and all potential alignments in the target speciestranscriptome. Extra weight was given to mismatches in the seed region,defined here as positions 2-9 of the antisense oligonucleotide, as wellthe cleavage site of the iRNA, defined here as positions 10-11 of theantisense oligonucleotide. The relative weight of the mismatches was2.8; 1.2:1 for seed mismatches, cleavage site, and other positions upthrough antisense position 19. Mismatches in the first position wereignored. A specificity score was calculated for each strand by summingthe value of each weighted mismatch. Preference was given to siRNAswhose antisense score in human and cynomolgus monkey was >=3.0 andpredicted efficacy was >=70% knockdown of the APOC3 transcript.

A series of iRNA duplexes with sequences designed as described abovewere synthesized, and conjugated with a trivalent GalNAc at the 3-end ofthe sense strand using the techniques described above. The sequences ofthese duplexes are shown in Tables 4A and 4B. These same sequences werealso synthesized with various nucleotide modifications and conjugatedwith a trivalent GalNAc. The sequences of the modified duplexes areshown in Table 5.

TABLE 4A APOC3 Unmodified Sequences Based on NM_000040.1. Posi- Posi-tion tion Sense Sense SEQ in Antisense Antisense SEQ in Duplex StrandSequence ID NM_000 Strand Sequence ID NM_00 ID ID (5′ to 3′) NO: 040.1ID (5′ to 3′) NO: 0040.1 AD- A- ACCAAGACCG 30 164-184 A- UGCAUCCUUG 133162-184 57501.1 117251.1 CCAAGGAUGC 117252.1 GCGGUCUUGG A UGG AD- A-ACCAAGACCG 31 164-184 A- UGCAUCCUUG 134 162-184 57537.1 117323.1CCAAGGAUGC 117324.1 GCGGUCUUGG A UGG AD- A- CAAGACCGCC 32 166-186 A-AGUGCAUCCU 135 164-186 57512.1 117271.1 AAGGAUGCAC 117272.1 UGGCGGUCUU UGGU AD- A- CAAGACCGCC 33 166-186 A- AGUGCAUCCU 136 164-186 57548.1117343.1 AAGGAUGCAC 117344.1 UGGCGGUCUU U GGU AD- A- CCGAUGGCUU 34237-257 A- UCAGGGAACU 137 235-257 57496.1 117249.1 CAGUUCCCUG 117250.1GAAGCCAUCG A GUC AD- A- CCGAUGGCUU 35 237-257 A- UCAGGGAACU 138 235-25757532.1 117321.1 CAGUUCCCUG 117322.1 GAAGCCAUCG A GUC AD- A- CGAUGGCUUC36 238-258 A- UUCAGGGAAC 139 236-258 57491.1 117247.1 AGUUCCCUGA117248.1 UGAAGCCAUC A GGU AD- A- CGAUGGCUUC 37 238-258 A- UUCAGGGAAC 140236-258 57527.1 117319.1 AGUUCCCUGA 117320.1 UGAAGCCAUC A GGU AD- A-AGACUACUGG 38 259-279 A- UUAACGGUGC 141 257-279 57547.1 117327.1AGCACCGUUA 117328.1 UCCAGUAGUC A UUU AD- A- AGACUACUGG 39 259-279 A-UUAACGGUGC 142 257-279 57511.1 117255.1 AGCACCGUUA 117256.1 UCCAGUAGUC AUUU AD- A- CUACUGGAGC 40 262-282 A- UCCUUAACGG 143 260-282 57561.1117365.1 ACCGUUAAGG 117366.1 UGCUCCAGUA A GUC AD- A- CUACUGGAGC 41262-282 A- UCCUUAACGG 144 260-282 57525.1 117293.1 ACCGUUAAGG 117294.1UGCUCCAGUA A GUC AD- A- ACUGGAGCAC 42 264-284 A- UGUCCUUAAC 145 262-28457520.1 117275.1 CGUUAAGGAC 117276.1 GGUGCUCCAG A UAG AD- A- ACUGGAGCAC43 264-284 A- UGUCCUUAAC 146 262-284 57556.1 117347.1 CGUUAAGGAC117348.1 GGUGCUCCAG A UAG AD- A- CUGGAGCACC 44 265-285 A- UUGUCCUUAA 147263-285 57503.1 117283.1 GUUAAGGACA 117284.1 CGGUGCUCCA A GUA AD- A-CUGGAGCACC 45 265-285 A- UUGUCCUUAA 148 263-285 57539.1 117355.1GUUAAGGACA 117356.1 CGGUGCUCCA A GUA AD- A- GGAGCACCGU 46 267-287 A-ACUUGUCCUU 149 265-287 57533.1 117337.1 UAAGGACAAG 117338.1 AACGGUGCUC UCAG AD- A- GGAGCACCGU 47 267-287 A- ACUUGUCCUU 150 265-287 57497.1117265.1 UAAGGACAAG 117266.1 AACGGUGCUC U CAG AD- A- GAGCACCGUU 48268-288 A- AACUUGUCCU 151 266-288 57498.1 117281.1 AAGGACAAGU 117282.1UAACGGUGCU U CCA AD- A- GAGCACCGUU 49 268-288 A- AACUUGUCCU 152 266-28857534.1 117353.1 AAGGACAAGU 117354.1 UAACGGUGCU U CCA AD- A- GUGGCUGCCU50 335-355 A- AUUGAGGUCU 153 333-355 57506.1 117253.1 GAGACCUCAA117254.1 CAGGCAGCCA U CGG AD- A- GUGGCUGCCU 51 335-355 A- AUUGAGGUCU 154333-355 57542.1 117325.1 GAGACCUCAA 117326.1 CAGGCAGCCA U CGG AD- A-GCCUGAGACC 52 341-361 A- UGGGGUAUUG 155 339-361 57523.1 117261.1UCAAUACCCC 117262.1 AGGUCUCAGG A CAG AD- A- GCCUGAGACC 53 341-361 A-UGGGGUAUUG 156 339-361 57559.1 117333.1 UCAAUACCCC 117334.1 AGGUCUCAGG ACAG AD- A- GGCUGCCUGA 54 337-357 A- GUAUUGAGGU 157 335-357 58915.1119685.1 GACCUCAAUA 119686.1 CUCAGGCAGC C CAC AD- A- CCUGAGACCU 55342-362 A- UUGGGGUAUU 158 340-362 57507.1 117269.1 CAAUACCCCA 117270.1GAGGUCUCAG A GCA AD- A- CCUGAGACCU 56 342-362 A- UUGGGGUAUU 159 340-36257543.1 117341.1 CAAUACCCCA 117342.1 GAGGUCUCAG A GCA AD- A- GCUGCCUGAG57 338-358 A- GGUAUUGAGG 160 336-358 58921.1 119690.1 ACCUCAAUAC119691.1 UCUCAGGCAG C CCA AD- A- CUGCCUGAGA 58 339-359 A- GGGUAUUGAG 161337-359 58913.1 119698.1 CCUCAAUACC 119699.1 GUCUCAGGCA C GCC AD- A-GACCUCAAUA 59 347-367 A- UGGACUUGGG 162 345-367 57502.1 117267.1CCCCAAGUCC 117268.1 GUAUUGAGGU A CUC AD- A- GACCUCAAUA 60 347-367 A-UGGACUUGGG 163 345-367 57538.1 117339.1 CCCCAAGUCC 117340.1 GUAUUGAGGU ACUC AD- A- UGAGACCUCA 61 344-364 A- ACUUGGGGUA 164 342-364 58923.1119692.1 AUACCCCAAG 119693.1 UUGAGGUCUC U AGG AD- A- AUCUCCAGGG 62405-425 A- UACAGGGGCA 165 403-425 58912.1 119683.1 CUGCCCCUGU 119684.1GCCCUGGAGA A UUG AD- A- GCUGCCCCUG 63 414-434 A- UAAGCAACCU 166 412-43457516.1 117273.1 UAGGUUGCUU 117274.1 ACAGGGGCAG A CCC AD- A- GCUGCCCCUG64 414-434 A- UAAGCAACCU 167 412-434 57552.1 117345.1 UAGGUUGCUU117346.1 ACAGGGGCAG A CCC AD- A- UGCCCCUGUA 65 416-436 A- UUUAAGCAAC 168414-436 57513.1 117287.1 GGUUGCUUAA 117288.1 CUACAGGGGC A AGC AD- A-UGCCCCUGUA 66 416-436 A- UUUAAGCAAC 169 414-436 57549.1 117359.1GGUUGCUUAA 117360.1 CUACAGGGGC A AGC AD- A- GCCCCUGUAG 67 417-437 A-UUUUAAGCAA 170 415-437 57519.1 117259.1 GUUGCUUAAA 117260.1 CCUACAGGGG ACAG AD- A- GCCCCUGUAG 68 417-437 A- UUUUAAGCAA 171 415-437 57555.1117331.1 GUUGCUUAAA 117332.1 CCUACAGGGG A CAG AD- A- CUGCCCCUGU 69415-435 A- UUAAGCAACC 172 413-435 58917.1 117367.2 AGGUUGCUUA 119687.1UACAGGGGCA A GCC AD- A- CUGUAGGUUG 70 421-441 A- UCCCUUUUAA 173 419-44157493.1 117279.1 CUUAAAAGGG 117280.1 GCAACCUACA A GGG AD- A- CUGUAGGUUG71 421-441 A- UCCCUUUUAA 174 419-441 57529.1 117351.1 CUUAAAAGGG117352.1 GCAACCUACA A GGG AD- A- CCCCUGUAGG 72 418-438 A- CUUUUAAGCA 175416-438 58914.1 119669.1 UUGCUUAAAA 119670.1 ACCUACAGGG G GCA AD- A-GUAGGUUGCU 73 423-443 A- UGUCCCUUUU 176 421-443 57521.1 117291.1UAAAAGGGAC 117292.1 AAGCAACCUA A CAG AD- A- GUAGGUUGCU 74 423-443 A-UGUCCCUUUU 177 421-443 57557.1 117363.1 UAAAAGGGAC 117364.1 AAGCAACCUA ACAG AD- A- CCCUGUAGGU 75 419-439 A- CCUUUUAAGC 178 417-439 58926.1119681.1 UGCUUAAAAG 119682.1 AACCUACAGG G GGC AD- A- GUUGCUUAAA 76427-447 A- AUACUGUCCC 179 425-447 57515.1 117257.1 AGGGACAGUA 117258.1UUUUAAGCAA U CCU AD- A- GUUGCUUAAA 77 427-447 A- AUACUGUCCC 180 425-44757551.1 117329.1 AGGGACAGUA 117330.1 UUUUAAGCAA U CCU AD- A- UUGCUUAAAA78 428-448 A- AAUACUGUCC 181 426-448 57544.1 117357.1 GGGACAGUAU117358.1 CUUUUAAGCA U ACC AD- A- UUGCUUAAAA 79 428-448 A- AAUACUGUCC 182426-448 57508.1 117285.1 GGGACAGUAU 117286.1 CUUUUAAGCA U ACC AD- A-GCUUAAAAGG 80 430-450 A- AGAAUACUGU 183 428-450 57517.1 117289.1GACAGUAUUC 117290.1 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 81 430-450 A-AGAAUACUGU 184 428-450 57553.1 117361.1 GACAGUAUUC 117362.1 CCCUUUUAAG UCAA AD- A- GCUUAAAAGG 82 430-450 A- AGAAUACUGU 185 428-450 64805.1129548.4 GACAGUAUUC 129547.4 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 83430-450 A- AGAAUACUGU 186 428-450 64793.1 117361.24 GACAGUAUUC 129547.3CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 84 430-450 A- AGAAUACUGU 187 428-45064799.1 129548.3 GACAGUAUUC 129546.18 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG85 430-450 A- AGAAUACUGU 188 428-450 64787.1 117361.23 GACAGUAUUC129546.17 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 86 430-450 A- AGAAUACUGU189 428-450 64813.1 117361.27 GACAGUAUUC 129565.2 CCCUUUUAAG U CAA AD-A- GCUUAAAAGG 87 430-450 A- AGAAUACUGU 190 428-450 64794.1 129554.4GACAGUAUUC 129546.24 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 88 430-450 A-AGAAUACUGU 191 428-450 64824.1 129559.2 GACAGUTUUC 129546.29 CCCUUUUAAGU CAA AD- A- GCUUAAAAGG 89 430-450 A- AGAAUACUGU 192 428-450 64825.1117361.29 GACAGUAUUC 129567.2 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 90430-450 A- AGAAUACUGU 193 428-450 64819.1 117361.28 GACAGUAUUC 129566.3CCCUUUUAAG U CAA AD- A- GCUUAAAAUG 91 430-450 A- AGAAUACUGU 194 428-45064828.1 129552.2 GACAGUAUUC 129546.22 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG92 430-450 A- AGAAUACUGU 195 428-450 64789.1 129561.2 GACAGUCUUC129546.31 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 93 430-450 A- AGAAUACUGU196 428-450 64807.1 117361.26 GACAGUAUUC 129564.3 CCCUUUUAAG U CAA AD-A- GCUUAAAAGG 94 430-450 A- AGAAUACUGU 197 428-450 64812.1 129557.4GACAGUTUUC 129546.27 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 95 430-450 A-AGAAUACUGU 198 428-450 64795.1 129562.2 GACAGUUUUC 129546.32 CCCUUUUAAGU CAA AD- A- GCUUAAAAGG 96 430-450 A- AGAAUACUGU 199 428-450 64804.1129554.6 GACAGUAUUC 129572.6 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 97430-450 A- AGAAUACUGU 200 428-450 64827.1 129550.8 GACAGUAUUC 129566.4CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 98 430-450 A- AGAAUACUGU 201 428-45064788.1 129553.5 GACAGTAUUC 129546.23 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG99 430-450 A- AGAAUACUGT 202 428-450 64832.1 129554.5 GACAGUAUUC129571.6 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 100 430-450 A- AGAAUACUGT203 428-450 64792.1 129553.6 GACAGTAUUC 129571.7 CCCTUUUAAG U CAA AD- A-GCUUAAAAGG 101 430-450 A- AGAAUACUGT 204 428-450 64831.1 117361.38GACAGUAUUC 129576.2 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 102 430-450 A-AGAAUACTGU 205 428-450 64820.1 117361.36 GACAGUAUUC 129574.2 CCCTUUUAAGU CAA AD- A- GCUUAAAAGG 103 430-450 A- AGAAUACUGU 206 428-450 64816.1129557.6 GACAGUTUUC 129572.8 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 104430-450 A- AGAAUACUGU 207 428-450 64811.1 129549.2 GACAGUAUUC 129546.19CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 105 430-450 A- AGAAUACUGU 208 428-45064821.1 129550.7 GACAGUAUUC 129564.4 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG106 430-450 A- AGAAUACUGU 209 428-450 64808.1 117361.34 GACAGUAUUC129572.5 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 107 430-450 A- AGAAUACUGU210 428-450 64810.1 129553.7 GACAGTAUUC 129572.7 CCCTUUUAAG U CAA AD- A-GCUUAAAAGG 108 430-450 A- AGAAUACUGU 211 428-450 64817.1 129550.5GACAGUAUUC 129546.20 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 109 430-450 A-AGAAUACUGU 212 428-450 64797.1 117361.40 GACAGUAUUC 129578.2 CCCUUUUAAGU CAA AD- A- GCUUAAAAGG 110 430-450 A- AGAAUACUGU 213 428-450 64829.1129560.2 GACAGUGUUC 129546.30 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG ill430-450 A- AGAAUACUGT 214 428-450 64802.1 117361.33 GACAGUAUUC 129571.5CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 112 430-450 A- AGAAUACUGT 215 428-45064798.1 129557.5 GACAGUTUUC 129571.8 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG113 430-450 A- AGAAUACUGU 216 428-450 64815.1 129550.6 GACAGUAUUC129581.2 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 114 430-450 A- AGAAUACUGT217 428-450 64791.1 117361.39 GACAGUAUUC 129577.2 CCCUUUUAAG U CAA AD-A- GCUUAAAAGG 115 430-450 A- AGAAUACUGU 218 428-450 64814.1 117361.35GACAGUAUUC 129573.2 CCCTUTUAAG U CAA AD- A- GCUUAAAAGG 116 430-450 A-AGAAUACUGU 219 428-450 64800.1 129555.2 GACAGUATUC 129546.25 CCCUUUUAAGU CAA AD- A- GCUUAAAAGG 117 430-450 A- AGAAUACUGU 220 428-450 64823.1129551.2 GACAGUAUUC 129546.21 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 118430-450 A- AGAAUACUGU 221 428-450 64818.1 129558.2 GACAGUAUUC 129546.28CCCUUUUAAG U CAA AD- A- GCUUAAAAGG 119 430-450 A- AGAAUACUGU 222 428-45064806.1 129556.2 GACAGUAUUC 129546.26 CCCUUUUAAG U CAA AD- A- GCUUAAAAGG120 430-450 A- AGAAUACUGT 223 428-450 64809.1 117361.42 GACAGUAUUC129580.3 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 121 430-450 A- AGAAUACUGT224 428-450 64822.1 129553.8 GACAGTAUUC 129580.4 CCCTUUUAAG U CAA AD- A-GCUUAAAAGG 122 430-450 A- AGAAUACUGU 225 428-450 64796.1 117361.32GACAGUAUUC 129570.2 CCCTUUUAAG U CAA AD- A- GCUUAAAAGG 123 430-450 A-AGAAUACUGT 226 428-450 64790.1 117361.31 GACAGUAUUC 129569.2 CCCTUUUAAGU CAA AD- A- CUUAAAAGGG 124 431-451 A- GAGAAUACUG 227 429-451 58920.1119675.1 ACAGUAUUCU 119676.1 UCCCUUUUAA C GCA AD- A- AAGGGACAGU 125436-456 A- GCACUGAGAA 228 434-456 58925.1 119694.1 AUUCUCAGUG 119695.1UACUGUCCCU C UUU AD- A- GGCCUCCCAA 126 499-519 A- GUCCAGCUUU 229 497-51958927.1 119696.1 UAAAGCUGGA 119697.1 AUUGGGAGGC C CAG AD- A- GCCUCCCAAU127 500-520 A- UGUCCAGCUU 230 498-520 58919.1 119688.1 AAAGCUGGAC119689.1 UAUUGGGAGG A CCA AD- A- CCUCCCAAUA 128 501-521 A- UUGUCCAGCU231 499-521 58924.1 119679.1 AAGCUGGACA 119680.1 UUAUUGGGAG A GCC AD- A-CUCCCAAUAA 129 502-522 A- CUUGUCCAGC 232 500-522 58916.1 119671.1AGCUGGACAA 119672.1 UUUAUUGGGA G GGC AD- A- UCCCAAUAAA 130 503-523 A-UCUUGUCCAG 233 501-523 58922.1 119677.1 GCUGGACAAG 119678.1 CUUUAUUGGG AAGG AD- A- CCCAAUAAAG 131 504-524 A- UUCUUGUCCA 234 502-524 58911.1119667.1 CUGGACAAGA 119668.1 GCUUUAUUGG A GAG AD- A- GCUGGACAAG 132513-533 A- CAUAGCAGCU 235 511-533 58918.1 119673.1 AAGCUGCUAU 119674.1UCUUGUCCAG G CUU

TABLE 4B APOC3 Unmodified Sequences Based on NM_023114.3 Posi- Posi-tion tion Sense SEQ in Antisense SEQ in Duplex Oligo Sense Sequence IDNM_023 Oligo Antisense Sequence ID NM_023 Name Name (5′ to 3′) NO: 114.3Name (5′ to 3′) NO: 114.3 AD- A- GGAUCCUUGCUGCUGGGCUC 236 125-145 A-AGAGCCCAGCAGCAAGGAUCCCU 288 123-145 57526.1 117309.1 U 117310.1 AD- A-GGAUCCUUGCUGCUGGGCUC 237 125-145 A- AGAGCCCAGCAGCAAGGAUCCCU 289 123-14557562.1 117381.1 U 117382.1 AD- A- GCUACAUGGAACAAGCCUCC 238 153-173 A-UGGAGGCUUGUUCCAUGUAGCCC 290 151-173 57504.1 117299.1 A 117300.1 AD- A-GCUACAUGGAACAAGCCUCC 239 153-173 A- UGGAGGCUUGUUCCAUGUAGCCC 291 151-17357540.1 117371.1 A 117372.1 AD- A- CUACAUGGAACAAGCCUCCA 240 154-174 A-UUGGAGGCUUGUUCCAUGUAGCC 292 152-174 57522.1 117307.1 A 117308.1 AD- A-CUACAUGGAACAAGCCUCCA 241 154-174 A- UUGGAGGCUUGUUCCAUGUAGCC 293 152-17457558.1 117379.1 A 117380.1 AD- A- ACAUGGAACAAGCCUCCAAG 242 156-176 A-UCUUGGAGGCUUGUUCCAUGUAG 294 154-176 57500.1 117313.1 A 117314.1 AD- A-ACAUGGAACAAGCCUCCAAG 243 156-176 A- UCUUGGAGGCUUGUUCCAUGUAG 295 154-17657536.1 117385.1 A 117386.1 AD- A- GGAACAAGCCUCCAAGACGG 244 160-180 A-ACCGUCUUGGAGGCUUGUUCCAU 296 158-180 57554.1 117377.1 U 117378.1 AD- A-GGAACAAGCCUCCAAGACGG 245 160-180 A- ACCGUCUUGGAGGCUUGUUCCAU 297 158-18057518.1 117305.1 U 117306.1 AD- A- ACAAGCCUCCAAGACGGUCC 246 163-183 A-UGGACCGUCUUGGAGGCUUGUUC 298 161-183 57545.1 117373.1 A 117374.1 AD- A-ACAAGCCUCCAAGACGGUCC 247 163-183 A- UGGACCGUCUUGGAGGCUUGUUC 299 161-18357509.1 117301.1 A 117302.1 AD- A- GCCUCCAAGACGGUCCAGGA 248 167-187 A-AUCCUGGACCGUCUUGGAGGCUU 300 165-187 57499.1 117297.1 U 117298.1 AD- A-GCCUCCAAGACGGUCCAGGA 249 167-187 A- AUCCUGGACCGUCUUGGAGGCUU 301 165-18757535.1 117369.1 U 117370.1 AD- A- CCCUGAAAGGCUACUGGAGC 250 258-278 A-UGCUCCAGUAGCCUUUCAGGGAU 302 256-278 57505.1 117315.1 A 117316.1 AD- A-CCCUGAAAGGCUACUGGAGC 251 258-278 A- UGCUCCAGUAGCCUUUCAGGGAU 303 256-27857541.1 117387.1 A 117388.1 AD- A- CCUGAAAGGCUACUGGAGCA 252 259-279 A-UUGCUCCAGUAGCCUUUCAGGGA 304 257-279 57510.1 117317.1 A 117318.1 AD- A-CCUGAAAGGCUACUGGAGCA 253 259-279 A- UUGCUCCAGUAGCCUUUCAGGGA 305 257-27957546.1 117389.1 A 117390.1 AD- A- UGAAAGGCUACUGGAGCAA 254 261-281 A-ACUUGCUCCAGUAGCCUUUCAGG 306 259-281 57495.1 117311.1 GU 117312.1 AD- A-UGAAAGGCUACUGGAGCAA 255 261-281 A- ACUUGCUCCAGUAGCCUUUCAGG 307 259-28157531.1 117383.1 GU 117384.1 AD- A- GAAAGGCUACUGGAGCAAG 256 262-282 A-AACUUGCUCCAGUAGCCUUUCAG 308 260-282 57514.1 117303.1 UU 117304.1 AD- A-GAAAGGCUACUGGAGCAAG 257 262-282 A- AACUUGCUCCAGUAGCCUUUCAG 309 260-28257550.1 117375.1 UU 117376.1 AD- A- AACAUGCUGUCCCUAAUAAA 258 348-368 A-CUUUAUUAGGGACAGCAUGUUUA 310 346-368 58954.1 119758.1 G 119759.1 AD- A-UAAGGGGAAAGUAUGUUCU 259 350-370 A- UGAGAACAUACUUUCCCCUUAAA 311 348-37058939.1 119720.1 CA 119721.1 AD- A- GCAGAUGUGCCUGUUCCUCC 260 352-372 A-UGGAGGAACAGGCACAUCUGCAA 312 350-372 58949.1 119740.1 A 119741.1 AD- A-AAGUAUGUUCUCAUGUCUU 261 367-387 A- UGAAGACAUGAGAACAUACUUUC 313 365-38758936.1 119750.1 CA 119751.1 AD- A- UCACCUAAACAUGCUGUCCC 262 373-393 A-AGGGACAGCAUGUUUAGGUGAGA 314 371-393 58940.1 119736.1 U 119737.1 AD- A-UUAAGGGGAAAGUAUGUUC 263 374-394 A- GAGAACAUACUUUCCCCUUAAAG 315 372-39458945.1 119738.1 UC 119739.1 AD- A- CCCUAGAUCUCACCUAAACA 264 377-397 A-AUGUUUAGGUGAGAUCUAGGGAG 316 375-397 58937.1 119766.1 U 119767.1 AD- A-UCCCUAAUAAAGCUGGAUA 265 393-413 A- CUUAUCCAGCUUUAUUAGGGACA 317 391-41358955.1 119712.1 AG 119713.1 AD- A- AAACAUGCUGUCCCUAAUAA 266 395-415 A-UUUAUUAGGGACAGCAUGUUUAG 318 393-415 58951.1 119710.1 A 119711.1 AD- A-CCCUAAUAAAGCUGGAUAA 267 409-429 A- UCUUAUCCAGCUUUAUUAGGGAC 319 407-42958947.1 119708.1 GA 119709.1 AD- A- CCUAAUAAAGCUGGAUAAG 268 416-436 A-UUCUUAUCCAGCUUUAUUAGGGA 320 414-436 58933.1 119702.1 AA 119703.1 AD- A-CUGAAGGUUGCUUUAAGGG 269 417-437 A- UCCCCUUAAAGCAACCUUCAGGG 321 415-43758931.1 119748.1 GA 119749.1 AD- A- AAGGGGAAAGUAUGUUCUC 270 423-443 A-AUGAGAACAUACUUUCCCCUUAA 322 421-443 58938.1 119704.1 AU 119705.1 AD- A-AGCUGGAUAAGAAGCUGCU 271 430-450 A- ACAGCAGCUUCUUAUCCAGCUUU 323 428-45058957.1 119744.1 GU 119745.1 AD- A- ACCUAAACAUGCUGUCCCUA 272 435-455 A-UUAGGGACAGCAUGUUUAGGUGA 324 433-455 58958.1 119760.1 A 119761.1 AD- A-UUUAAGGGGAAAGUAUGUU 273 452-472 A- AGAACAUACUUUCCCCUUAAAGC 325 450-47258930.1 119732.1 CU 119733.1 AD- A- UCGUGAGACUUCUGUGUUG 274 453-473 A-UGCAACACAGAAGUCUCACGACU 326 451-473 58932.1 119764.1 CA 119765.1 AD- A-AUUGAGUCGUGAGACUUCU 275 457-477 A- ACAGAAGUCUCACGACUCAAUAG 327 455-47758961.1 119746.1 GU 119747.1 AD- A- GUCCCUAAUAAAGCUGGAU 276 463-483 A-UUAUCCAGCUUUAUUAGGGACAG 328 461-483 58952.1 119726.1 AA 119727.1 AD- A-UUCUGUGUUGCAGAUGUGC 277 465-485 A- AGGCACAUCUGCAACACAGAAGU 329 463-48558946.1 119754.1 CU 119755.1 AD- A- UGGCCCCUGAAGGUUGCUUU 278 468-488 A-UAAAGCAACCUUCAGGGGCCACC 330 466-488 58956.1 119728.1 A 119729.1 AD- A-GUUGCUUUAAGGGGAAAGU 279 470-490 A- AUACUUUCCCCUUAAAGCAACCU 331 468-49058929.1 119716.1 AU 119717.1 AD- A- UGAGACUUCUGUGUUGCAG 280 473-493 A-AUCUGCAACACAGAAGUCUCACG 332 471-493 58948.1 119724.1 AU 119725.1 AD- A-GCUGGAUAAGAAGCUGCUG 281 475-495 A- AACAGCAGCUUCUUAUCCAGCUU 333 473-49558935.1 119734.1 UU 119735.1 AD- A- CUCCCUAGAUCUCACCUAAA 282 476-496 A-GUUUAGGUGAGAUCUAGGGAGGG 334 474-496 58944.1 119722.1 C 119723.1 AD- A-CCUAAACAUGCUGUCCCUAA 283 480-500 A- AUUAGGGACAGCAUGUUUAGGUG 335 478-50058959.1 119714.1 U 119715.1 AD- A- GAAAGUAUGUUCUCAUGUC 284 490-510 A-AAGACAUGAGAACAUACUUUCCC 336 488-510 58960.1 119730.1 UU 119731.1 AD- A-GCCCCUGAAGGUUGCUUUAA 285 497-517 A- CUUAAAGCAACCUUCAGGGGCCA 337 495-51758928.1 119700.1 G 119701.1 AD- A- CCCUCCCUAGAUCUCACCUA 286 501-521 A-UUAGGUGAGAUCUAGGGAGGGGU 338 499-521 58950.1 119756.1 A 119757.1 AD- A-CUGUCCCUAAUAAAGCUGGA 287 506-526 A- AUCCAGCUUUAUUAGGGACAGCA 339 504-52658962.1 119762.1 U 119763.1

TABLE 5 APOC3 Modified Sequences Sense Sense SEQ Antis Antisense SEQDuplex Oligo Sequence ID Oligo Sequence ID Name Name (5′ to 3′) NO: Name(5′ to 3′) NO: AD-57501.1 A-117251.1 AfcCfaAfgAfcCfGfCfcA 340 A-117252.1uGfcAfuCfcUfuGfgcgGf 495 faGfgAfuGfcAfL96 uCfuUfgGfusGfsg AD-57537.1A-117323.1 AfscsCfaAfgAfcCfGfCf 341 A-117324.1 usGfscAfuCfcUfuGfgcg 496cAfaGfgAfuGfcAfL96 GfuCfuUfgGfusGfsg AD-57512.1 A-117271.1CfaAfgAfcCfgCfCfAfaG 342 A-117272.1 aGfuGfcAfuCfcUfuggCf 497fgAfuGfcAfcUfL96 gGfuCfuUfgsGfsu AD-57548.1 A-117343.1CfsasAfgAfcCfgCfCfAf 343 A-117344.1 asGfsuGfcAfuCfcUfugg 498aGfgAfuGfcAfcUfL96 CfgGfuCfuUfgsGfsu AD-57496.1 A-117249.1CfcGfaUfgGfcUfUfCfaG 344 A-117250.1 uCfaGfgGfaAfcUfgaaGf 499fuUfcCfcUfgAfL96 cCfaUfcGfgsUfsc AD-57532.1 A-117321.1CfscsGfaUfgGfcUfUfCf 345 A-117322.1 usCfsaGfgGfaAfcUfgaa 500aGfuUfcCfcUfgAfL96 GfcCfaUfcGfgsUfsc AD-57491.1 A-117247.1CfgAfuGfgCfuUfCfAfgU 346 A-117248.1 uUfcAfgGfgAfaCfugaAf 501fuCfcCfuGfaAfL96 gCfcAfuCfgsGfsu AD-57527.1 A-117319.1CfsgsAfuGfgCfuUfCfAf 347 A-117320.1 usUfscAfgGfgAfaCfuga 502gUfuCfcCfuGfaAfL96 AfgCfcAfuCfgsGfsu AD-57547.1 A-117327.1AfsgsAfcUfaCfuGfGfAf 348 A-117328.1 usUfsaAfcGfgUfgCfucc 503gCfaCfcGfuUfaAfL96 AfgUfaGfuCfusUfsu AD-57511.1 A-117255.1AfgAfcUfaCfuGfGfAfgC 349 A-117256.1 uUfaAfcGfgUfgCfuccAf 504faCfcGfuUfaAfL96 gUfaGfuCfusUfsu AD-57561.1 A-117365.1CfsusAfcUfgGfaGfCfAf 350 A-117366.1 usCfscUfuAfaCfgGfugc 505cCfgUfuAfaGfgAfL96 UfcCfaGfuAfgsUfsc AD-57525.1 A-117293.1CfuAfcUfgGfaGfCfAfcC 351 A-117294.1 uCfcUfuAfaCfgGfugcUf 506fgUfuAfaGfgAfL96 cCfaGfuAfgsUfsc AD-57520.1 A-117275.1AfcUfgGfaGfcAfCfCfgU 352 A-117276.1 uGfuCfcUfuAfaCfgguGf 507fuAfaGfgAfcAfL96 cUfcCfaGfusAfsg AD-57556.1 A-117347.1AfscsUfgGfaGfcAfCfCf 353 A-117348.1 usGfsuCfcUfuAfaCfggu 508gUfuAfaGfgAfcAfL96 GfcUfcCfaGfusAfsg AD-57503.1 A-117283.1CfuGfgAfgCfaCfCfGfuU 354 A-117284.1 uUfgUfcCfuUfaAfcggUf 509faAfgGfaCfaAfL96 gCfuCfcAfgsUfsa AD-57539.1 A-117355.1CfsusGfgAfgCfaCfCfGf 355 A-117356.1 usUfsgUfcCfuUfaAfcgg 510uUfaAfgGfaCfaAfL96 UfgCfuCfcAfgsUfsa AD-57533.1 A-117337.1GfsgsAfgCfaCfcGfUfUf 356 A-117338.1 asCfsuUfgUfcCfuUfaac 511aAfgGfaCfaAfgUfL96 GfgUfgCfuCfcsAfsg AD-57497.1 A-117265.1GfgAfgCfaCfcGfUfUfaA 357 A-117266.1 aCfuUfgUfcCfuUfaacGf 512fgGfaCfaAfgUfL96 gUfgCfuCfcsAfsg AD-57498.1 A-117281.1GfaGfcAfcCfgUfUfAfaG 358 A-117282.1 aAfcUfuGfuCfcUfuaaCf 513fgAfcAfaGfuUfL96 gGfuGfcUfcsCfsa AD-57534.1 A-117353.1GfsasGfcAfcCfgUfUfAf 359 A-117354.1 asAfscUfuGfuCfcUfuaa 514aGfgAfcAfaGfuUfL96 CfgGfuGfcUfcsCfsa AD-57506.1 A-117253.1GfuGfgCfuGfcCfUfGfaG 360 A-117254.1 aUfuGfaGfgUfcUfcagGf 515faCfcUfcAfaUfL96 cAfgCfcAfcsGfsg AD-57542.1 A-117325.1GfsusGfgCfuGfcCfUfGf 361 A-117326.1 asUfsuGfaGfgUfcUfcag 516aGfaCfcUfcAfaUfL96 GfcAfgCfcAfcsGfsg AD-57523.1 A-117261.1GfcCfuGfaGfaCfCfUfcA 362 A-117262.1 uGfgGfgUfaUfuGfaggUf 517faUfaCfcCfcAfL96 cUfcAfgGfcsAfsg AD-57559.1 A-117333.1GfscsCfuGfaGfaCfCfUf 363 A-117334.1 usGfsgGfgUfaUfuGfagg 518cAfaUfaCfcCfcAfL96 UfcUfcAfgGfcsAfsg AD-58915.1 A-119685.1GfsgsCfuGfcCfuGfAfGf 364 A-119686.1 gsUfsaUfuGfaGfgUfcuc 519aCfcUfcAfaUfaCfL96 AfgGfcAfgCfcsasc AD-57507.1 A-117269.1CfcUfgAfgAfcCfUfCfaA 365 A-117270.1 uUfgGfgGfuAfuUfgagGf 520fuAfcCfcCfaAfL96 uCfuCfaGfgsCfsa AD-57543.1 A-117341.1CfscsUfgAfgAfcCfUfCf 366 A-117342.1 usUfsgGfgGfuAfuUfgag 521aAfuAfcCfcCfaAfL96 GfuCfuCfaGfgsCfsa AD-58921.1 A-119690.1GfscsUfgCfcUfgAfGfAf 367 A-119691.1 gsGfsuAfuUfgAfgGfucu 522cCfuCfaAfuAfcCfL96 CfaGfgCfaGfcscsa AD-58913.1 A-119698.1CfsusGfcCfuGfaGfAfCf 368 A-119699.1 gsGfsgUfaUfuGfaGfguc 523cUfcAfaUfaCfcCfL96 UfcAfgGfcAfgscsc AD-57502.1 A-117267.1GfaCfcUfcAfaUfAfCfcC 369 A-117268.1 uGfgAfcUfuGfgGfguaUf 524fcAfaGfuCfcAfL96 uGfaGfgUfcsUfsc AD-57538.1 A-117339.1GfsasCfcUfcAfaUfAfCf 370 A-117340.1 usGfsgAfcUfuGfgGfgua 525cCfcAfaGfuCfcAfL96 UfuGfaGfgUfcsUfsc AD-58923.1 A-119692.1UfsgsAfgAfcCfuCfAfAf 371 A-119693.1 asCfsuUfgGfgGfuAfuug 526uAfcCfcCfaAfgUfL96 AfgGfuCfuCfasgsg AD-58912.1 A-119683.1AfsusCfuCfcAfgGfGfCf 372 A-119684.1 usAfscAfgGfgGfcAfgcc 527uGfcCfcCfuGfuAfL96 CfuGfgAfgAfususg AD-57516.1 A-117273.1GfcUfgCfcCfcUfGfUfaG 373 A-117274.1 uAfaGfcAfaCfcUfacaGf 528fgUfuGfcUfuAfL96 gGfgCfaGfcsCfsc AD-57552.1 A-117345.1GfscsUfgCfcCfcUfGfUf 374 A-117346.1 usAfsaGfcAfaCfcUfaca 529aGfgUfuGfcUfuAfL96 GfgGfgCfaGfcsCfsc AD-57513.1 A-117287.1UfgCfcCfcUfgUfAfGfgU 375 A-117288.1 uUfuAfaGfcAfaCfcuaCf 530fuGfcUfuAfaAfL96 aGfgGfgCfasGfsc AD-57549.1 A-117359.1UfsgsCfcCfcUfgUfAfGf 376 A-117360.1 usUfsuAfaGfcAfaCfcua 531gUfuGfcUfuAfaAfL96 CfaGfgGfgCfasGfsc AD-57519.1 A-117259.1GfcCfcCfuGfuAfGfGfuU 377 A-117260.1 uUfuUfaAfgCfaAfccuAf 532fgCfuUfaAfaAfL96 cAfgGfgGfcsAfsg AD-57555.1 A-117331.1GfscsCfcCfuGfuAfGfGf 378 A-117332.1 usUfsuUfaAfgCfaAfccu 533uUfgCfuUfaAfaAfL96 AfcAfgGfgGfcsAfsg AD-58917.1 A-117367.2CfsusGfcCfcCfuGfUfAf 379 A-119687.1 usUfsaAfgCfaAfcCfuac 534gGfuUfgCfuUfaAfL96 AfgGfgGfcAfgscsc AD-57493.1 A-117279.1CfuGfuAfgGfuUfGfCfuU 380 A-117280.1 uCfcCfuUfuUfaAfgcaAf 535faAfaAfgGfgAfL96 cCfuAfcAfgsGfsg AD-57529.1 A-117351.1CfsusGfuAfgGfuUfGfCf 381 A-117352.1 usCfscCfuUfuUfaAfgca 536uUfaAfaAfgGfgAfL96 AfcCfuAfcAfgsGfsg AD-58914.1 A-119669.1CfscsCfcUfgUfaGfGfUf 382 A-119670.1 csUfsuUfuAfaGfcAfacc 537uGfcUfuAfaAfaGfL96 UfaCfaGfgGfgscsa AD-57521.1 A-117291.1GfuAfgGfuUfgCfUfUfaA 383 A-117292.1 uGfuCfcCfuUfuUfaagCf 538faAfgGfgAfcAfL96 aAfcCfuAfcsAfsg AD-57557.1 A-117363.1GfsusAfgGfuUfgCfUfUf 384 A-117364.1 usGfsuCfcCfuUfuUfaag 539aAfaAfgGfgAfcAfL96 CfaAfcCfuAfcsAfsg AD-58926.1 A-119681.1CfscsCfuGfuAfgGfUfUf 385 A-119682.1 csCfsuUfuUfaAfgCfaac 540gCfuUfaAfaAfgGfL96 CfuAfcAfgGfgsgsc AD-57515.1 A-117257.1GfuUfgCfuUfaAfAfAfgG 386 A-117258.1 aUfaCfuGfuCfcCfuuuUf 541fgAfcAfgUfaUfL96 aAfgCfaAfcsCfsu AD-57551.1 A-117329.1GfsusUfgCfuUfaAfAfAf 387 A-117330.1 asUfsaCfuGfuCfcCfuuu 542gGfgAfcAfgUfaUfL96 UfaAfgCfaAfcsCfsu AD-57544.1 A-117357.1UfsusGfcUfuAfaAfAfGf 388 A-117358.1 asAfsuAfcUfgUfcCfcuu 543gGfaCfaGfuAfuUfL96 UfuAfaGfcAfasCfsc AD-57508.1 A-117285.1UfuGfcUfuAfaAfAfGfgG 389 A-117286.1 aAfuAfcUfgUfcCfcuuUf 544faCfaGfuAfuUfL96 uAfaGfcAfasCfsc AD-57517.1 A-117289.1GfcUfuAfaAfaGfGfGfaC 390 A-117290.1 aGfaAfuAfcUfgUfcccUf 545faGfuAfuUfcUfL96 uUfuAfaGfcsAfsa AD-57553.1 A-117361.1GfscsUfuAfaAfaGfGfGf 391 A-117362.1 asGfsaAfuAfcUfgUfccc 546aCfaGfuAfuUfcUfL96 UfuUfuAfaGfcsAfsa AD-64805.1 A-129548.4Y44GfscsUfuAfaAfaGfG 392 A-129547.4 PasGfsaAfuAfcUfgUfcc 547fGfaCfaGfuAfuUfcUfL9 cUfuUfuAfaGfcsasa 6 AD-64793.1 A-117361.24GfscsUfuAfaAfaGfGfGf 393 A-129547.3 PasGfsaAfuAfcUfgUfcc 548aCfaGfuAfuUfcUfL96 cUfuUfuAfaGfcsasa AD-64799.1 A-129548.3Y44GfscsUfuAfaAfaGfG 394 A-129546.18 asGfsaAfuAfcUfgUfccc 549fGfaCfaGfuAfuUfcUfL9 UfuUfuAfaGfcsasa 6 AD-64787.1 A-117361.23GfscsUfuAfaAfaGfGfGf 395 A-129546.17 asGfsaAfuAfcUfgUfccc 550aCfaGfuAfuUfcUfL96 UfuUfuAfaGfcsasa AD-64813.1 A-117361.27GfscsUfuAfaAfaGfGfGf 396 A-129565.2 asGfsaauAfcUfgucccUf 551aCfaGfuAfuUfcUfL96 uuuaagcsasa AD-64794.1 A-129554.4gscsuuaaaaggdGacagu( 397 A-129546.24 asGfsaAfuAfcUfgUfccc 552Agn)uucuL96 UfuUfuAfaGfcsasa AD-64824.1 A-129559.2 gscsuuaaaaggdGdAcagu398 A-129546.29 asGfsaAfuAfcUfgUfccc 553 dTuucuL96 UfuUfuAfaGfcsasaAD-64825.1 A-117361.29 GfscsUfuAfaAfaGfGfGf 399 A-129567.2asGfsaauacugucccUfuu 554 aCfaGfuAfuUfcUfL96 uaagcsasa AD-64819.1A-117361.28 GfscsUfuAfaAfaGfGfGf 400 A-129566.3 asGfsaauAfcugucccUfu 555aCfaGfuAfuUfcUfL96 Ufuaagcsasa AD-64828.1 A-129552.2gscsuuaaaaugdGacagua 401 A-129546.22 asGfsaAfuAfcUfgUfccc 556 uucuL96UfuUfuAfaGfcsasa AD-64789.1 A-129561.2 gscsuuaaaaggdGacaguc 402A-129546.31 asGfsaAfuAfcUfgUfccc 557 uucuL96 UfuUfuAfaGfcsasa AD-64807.1A-117361.26 GfscsUfuAfaAfaGfGfGf 403 A-129564.3 asGfsaauAfcUfGfucccU 558aCfaGfuAfuUfcUfL96 fuUfuaagcsasa AD-64812.1 A-129557.4gscsuuaaaaggdGacagud 404 A-129546.27 asGfsaAfuAfcUfgUfccc 559 TuucuL96UfuUfuAfaGfcsasa AD-64795.1 A-129562.2 gscsuuaaaaggdGacaguu 405A-129546.32 asGfsaAfuAfcUfgUfccc 560 uucuL96 UfuUfuAfaGfcsasa AD-64804.1A-129554.6 gscsuuaaaaggdGacagu( 406 A-129572.6 asdGsaaudAcugucccdTu 561Agn)uucuL96 uuaagcsasa AD-64827.1 A-129550.8 gscsuuaaAfagGfGfacag 407A-129566.4 asGfsaauAfcugucccUfu 562 uauucuL96 Ufuaagcsasa AD-64788.1A-129553.5 gscsuuaaaaggdGacag(T 408 A-129546.23 asGfsaAfuAfcUfgUfccc 563gn)auucuL96 UfuUfuAfaGfcsasa AD-64832.1 A-129554.5 gscsuuaaaaggdGacagu(409 A-129571.6 asdGsaauacugdTcccdTu 564 Agn)uucuL96 uuaagcsasaAD-64792.1 A-129553.6 gscsuuaaaaggdGacag(T 410 A-129571.7asdGsaauacugdTcccdTu 565 gn)auucuL96 uuaagcsasa AD-64831.1 A-117361.38GfscsUfuAfaAfaGfGfGf 411 A-129576.2 asdGsaauacugdTcccUuu 566aCfaGfuAfuUfcUfL96 uaagcsasa AD-64820.1 A-117361.36 GfscsUfuAfaAfaGfGfGf412 A-129574.2 asdGsaaudAcdTgucccdT 567 aCfaGfuAfuUfcUfL96 uuuaagcsasaAD-64816.1 A-129557.6 gscsuuaaaaggdGacagud 413 A-129572.8asdGsaaudAcugucccdTu 568 TuucuL96 uuaagcsasa AD-64811.1 A-129549.2gscsuuAfaAfagGfGfaca 414 A-129546.19 asGfsaAfuAfcUfgUfccc 569 guauucuL96UfuUfuAfaGfcsasa AD-64821.1 A-129550.7 gscsuuaaAfagGfGfacag 415A-129564.4 asGfsaauAfcUfGfucccU 570 uauucuL96 fuUfuaagcsasa AD-64808.1A-117361.34 GfscsUfuAfaAfaGfGfGf 416 A-129572.5 asdGsaaudAcugucccdTu 571aCfaGfuAfuUfcUfL96 uuaagcsasa AD-64810.1 A-129553.7 gscsuuaaaaggdGacag(T417 A-129572.7 asdGsaaudAcugucccdTu 572 gn)auucuL96 uuaagcsasaAD-64817.1 A-129550.5 gscsuuaaAfagGfGfacag 418 A-129546.20asGfsaAfuAfcUfgUfccc 573 uauucuL96 UfuUfuAfaGfcsasa AD-64797.1A-117361.40 GfscsUfuAfaAfaGfGfGf 419 A-129578.2 asGsaaudAcugucccUuuu 574aCfaGfuAfuUfcUfL96 aagcsasa AD-64829.1 A-129560.2 gscsuuaaaaggdGacagug420 A-129546.30 asGfsaAfuAfcUfgUfccc 575 uucuL96 UfuUfuAfaGfcsasaAD-64802.1 A-117361.33 GfscsUfuAfaAfaGfGfGf 421 A-129571.5asdGsaauacugdTcccdTu 576 aCfaGfuAfuUfcUfL96 uuaagcsasa AD-64798.1A-129557.5 gscsuuaaaaggdGacagud 422 A-129571.8 asdGsaauacugdTcccdTu 577TuucuL96 uuaagcsasa AD-64815.1 A-129550.6 gscsuuaaAfagGfGfacag 423A-129581.2 asGfsaauAfcugUfcccUf 578 uauucuL96 uUfuaagcsasa AD-64791.1A-117361.39 GfscsUfuAfaAfaGfGfGf 424 A-129577.2 asGsaauacugdTcccUuuu 579aCfaGfuAfuUfcUfL96 aagcsasa AD-64814.1 A-117361.35 GfscsUfuAfaAfaGfGfGf425 A-129573.2 asdGsaauacugucccdTud 580 aCfaGfuAfuUfcUfL96 TuaagcsasaAD-64800.1 A-129555.2 gscsuuaaaaggdGacagua 426 A-129546.25asGfsaAfuAfcUfgUfccc 581 (Tgn)ucuL96 UfuUfuAfaGfcsasa AD-64823.1A-129551.2 gscsuuaaaaggdGacagua 427 A-129546.21 asGfsaAfuAfcUfgUfccc 582uucuL96 UfuUfuAfaGfcsasa AD-64818.1 A-129558.2 gscsuuaaaaggdGdAcagu 428A-129546.28 asGfsaAfuAfcUfgUfccc 583 dAuucuL96 UfuUfuAfaGfcsasaAD-64806.1 A-129556.2 gscsuuaaaaggdGacagud 429 A-129546.26asGfsaAfuAfcUfgUfccc 584 AuucuL96 UfuUfuAfaGfcsasa AD-64809.1A-117361.42 GfscsUfuAfaAfaGfGfGf 430 A-129580.3 PasgsaauacugdTcccdTu 585aCfaGfuAfuUfcUfL96 uuaagcsasa AD-64822.1 A-129553.8 gscsuuaaaaggdGacag(T431 A-129580.4 PasgsaauacugdTcccdTu 586 gn)auucuL96 uuaagcsasaAD-64796.1 A-117361.32 GfscsUfuAfaAfaGfGfGf 432 A-129570.2asgsaaudAcugucccdTuu 587 aCfaGfuAfuUfcUfL96 uaagcsasa AD-64790.1A-117361.31 GfscsUfuAfaAfaGfGfGf 433 A-129569.2 asgsaauacugdTcccdTuu 588aCfaGfuAfuUfcUfL96 uaagcsasa AD-58920.1 A-119675.1 CfsusUfaAfaAfgGfGfAf434 A-119676.1 gsAfsgAfaUfaCfuGfucc 589 cAfgUfaUfuCfuCfL96CfuUfuUfaAfgscsa AD-58925.1 A-119694.1 AfsasGfgGfaCfaGfUfAf 435A-119695.1 gsCfsaCfuGfaGfaAfuac 590 uUfcUfcAfgUfgCfL96 UfgUfcCfcUfususuAD-58927.1 A-119696.1 GfsgsCfcUfcCfcAfAfUf 436 A-119697.1gsUfscCfaGfcUfuUfauu 591 aAfaGfcUfgGfaCfL96 GfgGfaGfgCfcsasg AD-58919.1A-119688.1 GfscsCfuCfcCfaAfUfAf 437 A-119689.1 usGfsuCfcAfgCfuUfuau 592aAfgCfuGfgAfcAfL96 UfgGfgAfgGfcscsa AD-58924.1 A-119679.1CfscsUfcCfcAfaUfAfAf 438 A-119680.1 usUfsgUfcCfaGfcUfuua 593aGfcUfgGfaCfaAfL96 UfuGfgGfaGfgscsc AD-58916.1 A-119671.1CfsusCfcCfaAfuAfAfAf 439 A-119672.1 csUfsuGfuCfcAfgCfuuu 594gCfuGfgAfcAfaGfL96 AfuUfgGfgAfgsgsc AD-58922.1 A-119677.1UfscsCfcAfaUfaAfAfGf 440 A-119678.1 usCfsuUfgUfcCfaGfcuu 595cUfgGfaCfaAfgAfL96 UfaUfuGfgGfasgsg AD-58911.1 A-119667.1CfscsCfaAfuAfaAfGfCf 441 A-119668.1 usUfscUfuGfuCfcAfgcu 596uGfgAfcAfaGfaAfL96 UfuAfuUfgGfgsasg AD-58918.1 A-119673.1GfscsUfgGfaCfaAfGfAf 442 A-119674.1 csAfsuAfgCfaGfcUfucu 597aGfcUfgCfuAfuGfL96 UfgUfcCfaGfcsusu AD-57526.1 A-117309.1GfgAfuCfcUfuGfCfUfgC 443 A-117310.1 aGfaGfcCfcAfgCfagcAf 598fuGfgGfcUfcUfL96 aGfgAfuCfcsCfsu AD-57562.1 A-117381.1GfsgsAfuCfcUfuGfCfUf 444 A-117382.1 asGfsaGfcCfcAfgCfagc 599gCfuGfgGfcUfcUfL96 AfaGfgAfuCfcsCfsu AD-57504.1 A-117299.1GfcUfaCfaUfgGfAfAfcA 445 A-117300.1 uGfgAfgGfcUfuGfuucCf 600faGfcCfuCfcAfL96 aUfgUfaGfcsCfsc AD-57540.1 A-117371.1GfscsUfaCfaUfgGfAfAf 446 A-117372.1 usGfsgAfgGfcUfuGfuuc 601cAfaGfcCfuCfcAfL96 CfaUfgUfaGfcsCfsc AD-57522.1 A-117307.1CfuAfcAfuGfgAfAfCfaA 447 A-117308.1 uUfgGfaGfgCfuUfguuCf 602fgCfcUfcCfaAfL96 cAfuGfuAfgsCfsc AD-57558.1 A-117379.1CfsusAfcAfuGfgAfAfCf 448 A-117380.1 usUfsgGfaGfgCfuUfguu 603aAfgCfcUfcCfaAfL96 CfcAfuGfuAfgsCfsc AD-57500.1 A-117313.1AfcAfuGfgAfaCfAfAfgC 449 A-117314.1 uCfuUfgGfaGfgCfuugUf 604fcUfcCfaAfgAfL96 uCfcAfuGfusAfsg AD-57536.1 A-117385.1AfscsAfuGfgAfaCfAfAf 450 A-117386.1 usCfsuUfgGfaGfgCfuug 605gCfcUfcCfaAfgAfL96 UfuCfcAfuGfusAfsg AD-57554.1 A-117377.1GfsgsAfaCfaAfgCfCfUf 451 A-117378.1 asCfscGfuCfuUfgGfagg 606cCfaAfgAfcGfgUfL96 CfuUfgUfuCfcsAfsu AD-57518.1 A-117305.1GfgAfaCfaAfgCfCfUfcC 452 A-117306.1 aCfcGfuCfuUfgGfaggCf 607faAfgAfcGfgUfL96 uUfgUfuCfcsAfsu AD-57545.1 A-117373.1AfscsAfaGfcCfuCfCfAf 453 A-117374.1 usGfsgAfcCfgUfcUfugg 608aGfaCfgGfuCfcAfL96 AfgGfcUfuGfusUfsc AD-57509.1 A-117301.1AfcAfaGfcCfuCfCfAfaG 454 A-117302.1 uGfgAfcCfgUfcUfuggAf 609faCfgGfuCfcAfL96 gGfcUfuGfusUfsc AD-57499.1 A-117297.1GfcCfuCfcAfaGfAfCfgG 455 A-117298.1 aUfcCfuGfgAfcCfgucUf 610fuCfcAfgGfaUfL96 uGfgAfgGfcsUfsu AD-57535.1 A-117369.1GfscsCfuCfcAfaGfAfCf 456 A-117370.1 asUfscCfuGfgAfcCfguc 611gGfuCfcAfgGfaUfL96 UfuGfgAfgGfcsUfsu AD-57505.1 A-117315.1CfcCfuGfaAfaGfGfCfuA 457 A-117316.1 uGfcUfcCfaGfuAfgccUf 612fcUfgGfaGfcAfL96 uUfcAfgGfgsAfsu AD-57541.1 A-117387.1CfscsCfuGfaAfaGfGfCf 458 A-117388.1 usGfscUfcCfaGfuAfgcc 613uAfcUfgGfaGfcAfL96 UfuUfcAfgGfgsAfsu AD-57510.1 A-117317.1CfcUfgAfaAfgGfCfUfaC 459 A-117318.1 uUfgCfuCfcAfgUfagcCf 614fuGfgAfgCfaAfL96 uUfuCfaGfgsGfsa AD-57546.1 A-117389.1CfscsUfgAfaAfgGfCfUf 460 A-117390.1 usUfsgCfuCfcAfgUfagc 615aCfuGfgAfgCfaAfL96 CfuUfuCfaGfgsGfsa AD-57495.1 A-117311.1UfgAfaAfgGfcUfAfCfuG 461 A-117312.1 aCfuUfgCfuCfcAfguaGf 616fgAfgCfaAfgUfL96 cCfuUfuCfasGfsg AD-57531.1 A-117383.1UfsgsAfaAfgGfcUfAfCf 462 A-117384.1 asCfsuUfgCfuCfcAfgua 617uGfgAfgCfaAfgUfL96 GfcCfuUfuCfasGfsg AD-57514.1 A-117303.1GfaAfaGfgCfuAfCfUfgG 463 A-117304.1 aAfcUfuGfcUfcCfaguAf 618faGfcAfaGfuUfL96 gCfcUfuUfcsAfsg AD-57550.1 A-117375.1GfsasAfaGfgCfuAfCfUf 464 A-117376.1 asAfscUfuGfcUfcCfagu 619gGfaGfcAfaGfuUfL96 AfgCfcUfuUfcsAfsg AD-58954.1 A-119758.1AfsasCfaUfgCfuGfUfCf 465 A-119759.1 csUfsuUfaUfuAfgGfgac 620cCfuAfaUfaAfaGfL96 AfgCfaUfgUfususa AD-58939.1 A-119720.1UfsasAfgGfgGfaAfAfGf 466 A-119721.1 usGfsaGfaAfcAfuAfcuu 621uAfuGfuUfcUfcAfL96 UfcCfcCfuUfasasa AD-58949.1 A-119740.1GfscsAfgAfuGfuGfCfCf 467 A-119741.1 usGfsgAfgGfaAfcAfggc 622uGfuUfcCfuCfcAfL96 AfcAfuCfuGfcsasa AD-58936.1 A-119750.1AfsasGfuAfuGfuUfCfUf 468 A-119751.1 usGfsaAfgAfcAfuGfaga 623cAfuGfuCfuUfcAfL96 AfcAfuAfcUfususc AD-58940.1 A-119736.1UfscsAfcCfuAfaAfCfAf 469 A-119737.1 asGfsgGfaCfaGfcAfugu 624uGfcUfgUfcCfcUfL96 UfuAfgGfuGfasgsa AD-58945.1 A-119738.1UfsusAfaGfgGfgAfAfAf 470 A-119739.1 gsAfsgAfaCfaUfaCfuuu 625gUfaUfgUfuCfuCfL96 CfcCfcUfuAfasasg AD-58937.1 A-119766.1CfscsCfuAfgAfuCfUfCf 471 A-119767.1 asUfsgUfuUfaGfgUfgag 626aCfcUfaAfaCfaUfL96 AfuCfuAfgGfgsasg AD-58955.1 A-119712.1UfscsCfcUfaAfuAfAfAf 472 A-119713.1 csUfsuAfuCfcAfgCfuuu 627gCfuGfgAfuAfaGfL96 AfuUfaGfgGfascsa AD-58951.1 A-119710.1AfsasAfcAfuGfcUfGfUf 473 A-119711.1 usUfsuAfuUfaGfgGfaca 628cCfcUfaAfuAfaAfL96 GfcAfuGfuUfusasg AD-58947.1 A-119708.1CfscsCfuAfaUfaAfAfGf 474 A-119709.1 usCfsuUfaUfcCfaGfcuu 629cUfgGfaUfaAfgAfL96 UfaUfuAfgGfgsasc AD-58933.1 A-119702.1CfscsUfaAfuAfaAfGfCf 475 A-119703.1 usUfscUfuAfuCfcAfgcu 630uGfgAfuAfaGfaAfL96 UfuAfuUfaGfgsgsa AD-58931.1 A-119748.1CfsusGfaAfgGfuUfGfCf 476 A-119749.1 usCfscCfcUfuAfaAfgca 631uUfuAfaGfgGfgAfL96 AfcCfuUfcAfgsgsg AD-58938.1 A-119704.1AfsasGfgGfgAfaAfGfUf 477 A-119705.1 asUfsgAfgAfaCfaUfacu 632aUfgUfuCfuCfaUfL96 UfuCfcCfcUfusasa AD-58957.1 A-119744.1AfsgsCfuGfgAfuAfAfGf 478 A-119745.1 asCfsaGfcAfgCfuUfcuu 633aAfgCfuGfcUfgUfL96 AfuCfcAfgCfususu AD-58958.1 A-119760.1AfscsCfuAfaAfcAfUfGf 479 A-119761.1 usUfsaGfgGfaCfaGfcau 634cUfgUfcCfcUfaAfL96 GfuUfuAfgGfusgsa AD-58930.1 A-119732.1UfsusUfaAfgGfgGfAfAf 480 A-119733.1 asGfsaAfcAfuAfcUfuuc 635aGfuAfuGfuUfcUfL96 CfcCfuUfaAfasgsc AD-58932.1 A-119764.1UfscsGfuGfaGfaCfUfUf 481 A-119765.1 usGfscAfaCfaCfaGfaag 636cUfgUfgUfuGfcAfL96 UfcUfcAfcGfascsu AD-58961.1 A-119746.1AfsusUfgAfgUfcGfUfGf 482 A-119747.1 asCfsaGfaAfgUfcUfcac 637aGfaCfuUfcUfgUfL96 GfaCfuCfaAfusasg AD-58952.1 A-119726.1GfsusCfcCfuAfaUfAfAf 483 A-119727.1 usUfsaUfcCfaGfcUfuua 638aGfcUfgGfaUfaAfL96 UfuAfgGfgAfcsasg AD-58946.1 A-119754.1UfsusCfuGfuGfuUfGfCf 484 A-119755.1 asGfsgCfaCfaUfcUfgca 639aGfaUfgUfgCfcUfL96 AfcAfcAfgAfasgsu AD-58956.1 A-119728.1UfsgsGfcCfcCfuGfAfAf 485 A-119729.1 usAfsaAfgCfaAfcCfuuc 640gGfuUfgCfuUfuAfL96 AfgGfgGfcCfascsc AD-58929.1 A-119716.1GfsusUfgCfuUfuAfAfGf 486 A-119717.1 asUfsaCfuUfuCfcCfcuu 641gGfgAfaAfgUfaUfL96 AfaAfgCfaAfcscsu AD-58948.1 A-119724.1UfsgsAfgAfcUfuCfUfGf 487 A-119725.1 asUfscUfgCfaAfcAfcag 642uGfuUfgCfaGfaUfL96 AfaGfuCfuCfascsg AD-58935.1 A-119734.1GfscsUfgGfaUfaAfGfAf 488 A-119735.1 asAfscAfgCfaGfcUfucu 643aGfcUfgCfuGfuUfL96 UfaUfcCfaGfcsusu AD-58944.1 A-119722.1CfsusCfcCfuAfgAfUfCf 489 A-119723.1 gsUfsuUfaGfgUfgAfgau 644uCfaCfcUfaAfaCfL96 CfuAfgGfgAfgsgsg AD-58959.1 A-119714.1CfscsUfaAfaCfaUfGfCf 490 A-119715.1 asUfsuAfgGfgAfcAfgca 645uGfuCfcCfuAfaUfL96 UfgUfuUfaGfgsusg AD-58960.1 A-119730.1GfsasAfaGfuAfuGfUfUf 491 A-119731.1 asAfsgAfcAfuGfaGfaac 646cUfcAfuGfuCfuUfL96 AfuAfcUfuUfcscsc AD-58928.1 A-119700.1GfscsCfcCfuGfaAfGfGf 492 A-119701.1 csUfsuAfaAfgCfaAfccu 647uUfgCfuUfuAfaGfL96 UfcAfgGfgGfcscsa AD-58950.1 A-119756.1CfscsCfuCfcCfuAfGfAf 493 A-119757.1 usUfsaGfgUfgAfgAfucu 648uCfuCfaCfcUfaAfL96 AfgGfgAfgGfgsgsu AD-58962.1 A-119762.1CfsusGfuCfcCfuAfAfUf 494 A-119763.1 asUfscCfaGfcUfuUfauu 649aAfaGfcUfgGfaUfL96 AfgGfgAfcAfgscsa

Example 2. In Vitro Testing of siRNA Sequences

Table 6 shows the results of a single dose screen in Hep3B cells usingthe selected modified APOC3 iRNAs. The data are expressed as percent ofAPOC3 mRNA remaining in the cells transfected with iRNAs relative toAPOC3 mRNA remaining in the cells transfected with the AD-1955non-targeting control.

TABLE 6 Results of APOC3 single dose screen. Avg. Avg. SD SD Duplex Name10 nM 0.1 nM 10 nM 0.1 nM AD-57517.1 1.2 3.9 0.8 3.2 AD-57544.1 2.4 18.21.1 14.2 AD-57515.1 2.5 7.4 2.0 6.0 AD-57551.1 2.8 19.4 2.2 14.8AD-57553.1 3.0 11.2 2.1 10.7 AD-57498.1 3.4 19.3 2.4 16.0 AD-57508.1 6.111.8 7.8 7.8 AD-57523.1 6.2 39.7 1.2 20.8 AD-57519.1 7.7 29.5 1.1 17.9AD-57561.1 8.0 50.7 2.3 31.0 AD-57502.1 9.0 27.2 2.0 14.6 AD-57547.1 9.839.4 4.3 21.9 AD-57511.1 10.6 31.0 3.0 19.6 AD-57493.1 10.9 42.0 6.022.1 AD-57555.1 11.1 42.0 5.7 25.3 AD-57503.1 12.9 36.9 3.2 23.5AD-57496.1 13.6 56.3 4.0 20.9 AD-57559.1 13.7 66.1 5.7 37.5 AD-57513.114.2 46.7 4.8 31.5 AD-57534.1 15.5 46.5 17.3 23.7 AD-57491.1 15.6 35.66.8 14.6 AD-57512.1 15.8 48.0 5.7 29.6 AD-57525.1 16.5 24.7 19.7 14.7AD-57539.1 17.8 54.8 10.7 21.0 AD-57538.1 20.1 57.8 13.2 26.3 AD-57529.120.5 62.3 9.7 29.7 AD-57527.1 22.5 55.4 13.3 29.8 AD-57521.1 25.8 42.224.4 22.2 AD-57501.1 25.9 66.6 8.4 25.3 AD-57548.1 26.1 63.0 12.2 27.2AD-57516.1 29.2 55.2 10.4 29.8 AD-57533.1 29.7 58.9 22.8 29.8 AD-57532.130.4 62.2 29.3 18.0 AD-57497.1 30.5 57.2 6.0 31.2 AD-57549.1 31.8 57.216.3 34.2 AD-57506.1 34.3 55.5 9.0 30.7 AD-57557.1 35.3 62.9 22.0 27.7AD-57520.1 37.6 52.7 17.7 26.5 AD-57556.1 38.1 59.9 18.3 25.7 AD-57505.139.8 63.2 19.2 18.5 AD-57542.1 41.4 60.0 6.0 24.4 AD-57552.1 41.4 62.813.3 25.8 AD-57537.1 45.4 61.7 20.4 22.4 AD-57541.1 58.3 72.5 13.8 26.0AD-57495.1 59.5 68.5 5.2 27.7 AD-57507.1 64.2 62.0 2.4 33.1 AD-57510.167.2 62.2 0.4 25.1 AD-57522.1 67.5 67.4 15.5 30.6 AD-57504.1 69.1 68.17.8 23.3 AD-57546.1 72.1 69.8 9.6 30.4 AD-57543.1 73.0 68.5 0.1 29.1AD-57558.1 75.6 68.3 11.9 27.2 AD-57545.1 82.5 66.4 1.6 24.4 AD-57509.183.4 71.2 2.1 31.0 AD-57514.1 85.2 69.9 2.8 29.2 AD-57550.1 85.2 64.33.0 19.0 AD-57540.1 86.1 67.3 11.5 27.7 AD-57500.1 86.3 73.3 2.7 31.3AD-57499.1 89.1 73.9 2.2 28.2 AD-57536.1 90.1 75.6 13.4 35.8 AD-57554.193.0 67.1 0.2 27.3 AD-57518.1 95.0 68.0 1.5 21.7 AD-57526.1 96.5 88.53.9 38.7 AD-57531.1 99.8 73.6 28.0 30.1 AD-57535.1 101.3 72.0 12.9 32.7AD-57562.1 103.6 81.2 8.6 38.6 AD-58925.1 11.0 32.2 0.6 1.1 AD-58911.112.1 25.2 0.4 0.9 AD-58924.1 14.0 26.9 0.8 2.3 AD-58933.1 15.1 43.8 0.41.1 AD-58922.1 15.3 25.0 1.0 1.8 AD-58916.1 23.0 57.6 0.9 0.5 AD-58935.123.3 59.5 0.4 ND AD-58920.1 23.6 59.5 0.6 ND AD-58918.1 26.6 57.3 0.61.2 AD-58917.1 29.4 71.9 1.1 3.5 AD-58914.1 31.0 58.5 1.3 3.4 AD-58919.131.8 83.8 1.9 1.6 AD-58913.1 32.4 68.6 1.5 2.1 AD-58957.1 36.0 81.2 1.40.8 AD-58923.1 42.8 86.5 2.9 1.5 AD-58915.1 48.4 77.7 0.8 2.6 AD-58927.157.0 92.6 1.6 1.4 AD-58962.1 57.9 96.7 5.5 3.7 AD-58921.1 63.9 95.8 2.52.8 AD-58926.1 65.1 88.1 2.0 2.5 AD-58947.1 69.4 93.6 3.4 3.8 AD-58928.174.5 91.3 5.3 0.0 AD-58931.1 77.2 96.6 6.2 2.0 AD-58950.1 82.4 98.3 1.11.0 AD-58936.1 89.2 102.0 2.3 1.3 AD-58956.1 93.0 102.4 1.4 NDAD-58912.1 93.9 98.0 3.1 3.4 AD-58951.1 97.0 103.5 2.7 1.4 AD-58949.197.3 110.5 1.8 ND AD-58955.1 97.4 103.8 3.7 1.3 AD-58948.1 97.9 101.67.9 3.6 AD-58937.1 98.7 89.4 4.8 2.2 AD-58932.1 99.4 97.3 5.8 NDAD-58946.1 100.8 97.9 2.0 ND AD-58944.1 106.5 83.9 4.7 4.2 AD-58954.1108.7 95.1 3.5 1.5 AD-58940.1 109.0 97.7 ND 0.3 AD-58961.1 110.4 111.3ND 6.0 AD-58952.1 110.7 96.0 ND 1.5 AD-58945.1 114.5 101.5 ND 1.5AD-58929.1 115.0 102.9 ND ND AD-58958.1 115.7 118.1 ND ND AD-58938.1118.5 92.4 ND 2.7 AD-58939.1 119.3 98.7 ND 0.8 AD-58960.1 120.6 101.2 NDND AD-58930.1 123.7 108.6 ND 1.2 AD-58959.1 133.2 123.3 ND 3.2AD-64805.1 4.4 18.8 2.2 11.0 AD-64793.1 6.1 23.9 3.2 9.6 AD-64799.1 6.051.4 3.3 10.8 AD-64787.1 8.2 45.0 3.8 24.0 AD-64813.1 9.1 45.5 4.9 16.0AD-64794.1 8.7 50.2 2.1 15.9 AD-64824.1 9.6 57.5 2.2 5.4 AD-64825.1 12.760.3 8.9 14.2 AD-64819.1 12.2 62.4 5.1 13.6 AD-64828.1 16.9 49.2 6.5 6.7AD-64789.1 21.8 39.7 6.1 5.7 AD-64807.1 20.0 48.7 9.8 17.5 AD-64812.118.2 59.3 6.1 19.9 AD-64795.1 19.9 54.2 6.3 10.1 AD-64804.1 18.0 59.46.0 7.6 AD-64827.1 17.3 67.3 8.8 17.7 AD-64788.1 15.9 69.2 3.4 11.6AD-64832.1 19.0 61.4 6.8 7.5 AD-64792.1 24.6 54.6 7.6 6.2 AD-64831.120.7 63.8 4.2 9.0 AD-64820.1 19.6 73.9 7.7 7.2 AD-64816.1 26.7 52.8 6.16.5 AD-64811.1 15.4 97.5 4.2 17.6 AD-64821.1 18.7 79.8 4.6 11.5AD-64808.1 27.6 58.4 12.0 8.9 AD-64810.1 30.8 55.7 10.1 7.2 AD-64817.120.0 91.2 9.1 8.4 AD-64797.1 26.1 67.1 3.2 15.3 AD-64829.1 25.5 75.810.3 18.4 AD-64802.1 34.2 63.9 11.0 10.1 AD-64798.1 34.5 66.9 18.0 8.3AD-64815.1 29.3 75.2 6.0 12.4 AD-64791.1 38.6 72.1 7.6 13.5 AD-64814.135.7 87.6 14.9 11.8 AD-64800.1 37.9 96.0 10.2 13.8 AD-64823.1 33.1 110.57.7 9.3 AD-64818.1 41.3 120.3 8.0 10.5 AD-64806.1 52.1 103.8 13.8 28.7AD-64809.1 84.7 81.4 16.3 22.4 AD-64822.1 98.0 89.6 26.0 6.2 AD-64796.1103.4 95.4 20.8 10.5 AD-64790.1 131.8 84.5 46.9 8.5

Table 7 shows the dose response of Hep3B cells transfected with theindicated cyno/human cross reactive modified APOC3 iRNAs. The indicatedIC₅₀ values represent the IC₅₀ values relative to untreated cells. Theresults of a single dose screen (Avg. 10 nM blue bars and Avg. 0.1 nMred bars) for APOC3 iRNAs are shown in FIG. 1 . Based on the results ofthe screen, three iRNAs (AD-57553.1, AD-57547.1 and AD-58924.1) wereselected for further in vivo testing.

TABLE 7 APOC3 Dose Response Screen IC50 Duplex ID (nM) AD-57553.1 0.012AD-57544.1 0.022 AD-57551.1 0.021 AD-57547.1 0.044 AD-57555.1 0.075AD-57534.1 0.142 AD-57549.1 0.871 AD-57527.1 0.209 AD-57533.1 1.425AD-57538.1 0.177 AD-57559.1 0.395 AD-58925.1 0.13 AD-58911.1 0.09AD-58924.1 0.08 AD-58933.1 0.39 AD-58922.1 0.09 AD-58916.1 0.40AD-58935.1 0.62 AD-58920.1 0.30 AD-58918.1 0.97 AD-58917.1 1.78AD-58914.1 3.20

Example 3. In Vivo Testing of AD-57558 siRNA Sequence in Wild-Type Mice

The rodent-specific AD-57558-GalNAc3 sequence was tested in wild-typemice for its ability to inhibit the expression of APOC3 and to reduceserum lipids. AD-57558-GalNAc3 was administered subcutaneously at 3mg/kg, 10 mg/kg or 30 mg/kg as a single dose, with PBS used as anegative control. The expression of APOC3 was measured 5 days afterdosing by RT-PCR. The results, shown in FIG. 2 , indicate thatAD-57558-GalNAc3 is able to reduce the expression of APOC3 by about 60%(for a 3 mg/kg dose), about 80% (for a 10 mg/kg dose) and about 85% (fora 30 mg/kg dose).

Example 4. Generation and Characterization of a Mouse Model of APOC3Overexpression

To enable ApoC3 GalNAc conjugate lead finding in vivo with compoundsthat do not cross-react with the rodent APOC3 gene, a system foroverexpression of the human APOC3 gene in the liver of C57Bl/6 mice wasemployed. An adeno-associated virus serotype 8 (AAV8) encapsidated AAV2vector genome expressing human APOC3 under the liver-specific TBGpromoter was generated. Model characterization studies were performed toidentify optimal conditions, including the number of viral genome copies(GC) and time needed for high, durable expression of human ApoC3 inAAV-transduced mice.

Further testing was carried out using the dosing of 10¹¹ genome copiesof the AAV2 vector per mouse. The levels of APOC3 mRNA in the liver ofAAV-hApoC3 mice were measured by RT-PCR 1.5 weeks, 6.5 weeks, 8 weeksand 16 weeks after administration of the hAPOC3 AAV vector. The resultsof RT-PCR (data not shown) indicate that high expression levels of thehuman APOC3 gene were achieved in mice within 10 days followingadministration of 10¹¹ genome copies of hAPOC3 AAV and were sustainedfor at least 6 months with little animal-to-animal variability.

Example 5. Testing Potential Lead iRNAs in a Mouse Model of APOC3Overexpression

Once characterized, the APOC3-AAV mouse model system of APOC3overexpression was used for in vivo screening of AD-57553, AD-57547 andAD-58924 that were identified based on their potency in vitro. Forsingle high dose screen experiments, APOC3-AAV mice previously injectedwith 10¹¹ genome copies of hAPOC3 AAV were administered a single 10mg/kg dose of AD-57553, AD-57547 and AD-58924 or PBS (as control), andAPOC3 mRNA was subsequently measured. The results are presented in FIGS.3 and 4 . Specifically, FIG. 3 shows the levels of APOC3 mRNA measuredin individual APOC3-AAV mice injected with AD-57553, AD-57547 andAD-58924 or PBS, while FIG. 4 shows group mRNA averages. The dataindicate that AD-57553 is the most effective in inhibiting APOC3expression.

AD-57553 was selected for further in vivo testing in dose response andmulti-dose studies. For the dose response experiments, APOC3-AAV micepreviously injected with 10¹¹ genome copies of hAPOC3 AAV wereadministered 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg doses weekly for 4 weeks,and the APOC3 mRNA levels were subsequently measured. The results arepresented in FIGS. 5 and 6 . Specifically, FIG. 5 shows the levels ofAPOC3 mRNA measured in individual APOC3-AAV mice injected with AD-57553,while FIG. 6 shows group mRNA averages. The data show an increasinginhibition of APOC3 mRNA expression with the increasing dose ofAD-57553.

Example 6. Generation and In Vivo Testing of Additional iRNA SequencesBased on AD-57553

From the initial round of SAR optimization of iRNA chemistry, 10additional iRNA sequences were generated based on the AD-57553 leadsequence by introducing changes in 2′F, 2′OMe and 5′P modifications. Theadditional iRNAs are presented in Tables 8 and 9 below.

TABLE 8 Additional iRNA Sequences (Unmodified) Sense Anti- AntisenseSense Sequence SEQ sense Sequence SEQ Duplex Oligo (5′ to ID Oligo(5′ to ID Name Name 3′) NO: Name 3′) NO: AD- A- GCUUAAAA 650 A- AGAAUACU661 57553.3 117361.2 GGGACAGU 117362.2 GUCCCUUU AUUCU UAA GCAA AD- A-GCUUAAAA 651 A- UGAAUACU 662 65696.1 117361.2 GGGACAGU 130731.1 GUCCCUUUAUUCU UAA GCAA AD- A- GCUUAAAA 652 A- UGAAUACU 663 65697.1 130732.1GGGACAGU 130734.1 GUCCCUUU AUUCA UAA GCAA AD- A- GCUUAAAA 653 A-UGAAUACU 664 65698.1 130732.1 GGGACAGU 130735.1 GUCCCUUU AUUCA UAA GCAAAD- A- GCUUAAAA 654 A- UGAAUACU 665 65699.1 130732.1 GGGACAGU 130736.1GTCCCTUU AUUCA UAA GCAA AD- A- GCUUAAAA 655 A- UGAAUACU 666 65700.1130733.1 GGGACAGU 130734.1 GUCCCUUU CUUCA UAA GCAA AD- A- GCUUAAAA 656A- UGAAUACU 667 65701.1 130733.1 GGGACAGU 130735.1 GUCCCUUU CUUCA UAAGCAA AD- A- GCUUAAAA 657 A- UGAAUACU 668 65702.1 130733.1 GGGACAGU130736.1 GTCCCTUU CUUCA UAA GCAA AD- A- GCUUAAAA 658 A- UGAAUACU 66965703.1 130737.1 GGGACAGU 130734.1 GUCCCUUU AUUCA UAA GCAA AD- A-GCUUAAAA 659 A- UGAAUACU 670 65704.1 130737.1 GGGACAGU 130735.1 GUCCCUUUAUUCA UAA GCAA AD- A- GCUUAAAA 660 A- UGAAUACU 671 65705.1 130737.1GGGACAGU 130736.1 GTCCCTUU AUUCA UAA GCAA

TABLE 9 Additional iRNA Sequences (Modified) Sense Anti- Antisense SenseSequence SEQ sense Sequence SEQ Duplex Oligo (5′ to ID Oligo (5′ to IDName Name 3′) NO: Name 3′) NO: AD- A- GfscsUfu 672 A- asGfsaAf 68357553.3 117361.2 AfaAfaGf 117362.2 uAfcUfgU GfGfaCfa fcccUfuU Gfu fuAfaAfuUfcUf GfcsAfsa L96 AD- A- GfscsUfu 673 A- VPusGfsa 684 65696.1117361.2 AfaAfaGf 130731.1 AfuAfcUf GfGfaCfa gUfcccUf Gfu uUfu AfuUfcUfAfaGfesa L96 sa AD- A- gscsuuaa 674 A- usGfsaau 685 65697.1 130732.1aaggdGac 130734.1 AfcUfGfu agu(Agn) cccUfuUf uuca uaagcs L96 asa AD- A-gscsuuaa 675 A- usGfsaau 686 65698.1 130732.1 aaggdGac 130735.1 acugucccagu(Agn) Ufuuuaag uuca csasa L96 AD- A- gscsuuaa 676 A- usdGsaau 68765699.1 130732.1 aaggdGac 130736.1 acugdTcc agu(Agn) cdTuuuaa uucagcsasa L96 AD- A- gscsuuaa 677 A- usGfsaau 688 65700.1 130733.1 aaggdGac130734.1 AfcUfGfu agucuuca cccUfuUf L96 uaagcs asa AD- A- gscsuuaa 678A- usGfsaau 689 65701.1 130733.1 aaggdGac 130735.1 acuguccc agucuucaUfuuuaag L96 csasa AD- A- gscsuuaa 679 A- usdGsaau 690 65702.1 130733.1aaggdGac 130736.1 acugdTcc agucuuca cdTuuuaa L96 gcsasa AD- A- gscsuuaa680 A- usGfsaau 691 65703.1 130737.1 AfaGfGfG 130734.1 AfcUfGfu facaguaucccUfuUf ucaL uaagcs 96 asa AD- A- gscsuuaa 681 A- usGfsaau 692 65704.1130737.1 AfaGfGfG 130735.1 acuguccc facaguau Ufuuuaag ucaL csasa 96 AD-A- gscsuuaa 682 A- usdGsaau 693 65705.1 130737.1 AfaGfGfG 130736.1acugdTcc facaguau cdTuuuaa ucaL gcsasa 96

The additional sequences were tested in APOC3-AAV mice for their abilityto inhibit the expression of the APOC3 protein. Specifically, APOC3-AAVmice previously injected with 10¹¹ genome copies of hAPOC3 AAV wereadministered a single dose of 3 mg/kg of the indicated modified iRNA,and the resulting levels of circulating serum APOC3 protein weremeasured on days 5, 10 and 20 after dosing. FIG. 7A presents the timecourse for up to 20 days for the levels of serum APOC3 protein measuredfor each tested iRNA sequence and FIG. 7B presents the time course forup to 30 days for the levels of serum APOC3 protein for six selectediRNA sequences. FIGS. 8 and 9 represent the data for each tested iRNAsequence for days 10 and 20, respectively. The data indicate that themost active iRNA sequences, such as AD-65704, are able to achieve about80% knockdown of serum APOC3 protein on days 10 and 20.

Example 7. Testing New Lead iRNAs in a Mouse Model of APOC3Overexpression

AD-57553, AD-65696, AD-65703 and AD-65704 were selected for follow-onstudies in order to test the effect of fluorine content and of vinylphosphate on the ability of the iRNA agent to inhibit the expression ofAPOC3 protein in vivo. Table 9 above shows the modified sequences ofAD-57553, AD-65696, AD-65703 and AD-65704, and Table 10 below contains abrief explanation of the modifications present in each strand.

TABLE 10  Modified Sequences of Selected iRNAsUsed for In Vivo Experiments Anti- Sense sense Se- Se- Explanationquence SEQ quence SEQ of Duplex (5′ to ID (5′ to ID Modifi- Name 3′) NO:3′) NO: cations AD- Gfscs 694 asGfs 700 Contains 6 57553.3 UfuAf aAfuAphosphoro- aAfaG fcUfg thioates fGfGf Ufccc a U CfaGf fuUfu uAfuU AfaGffcUfL csAfs 96 a AD- Gfscs 695 VPusG 701 Contains 6 65696.1 UfuAf fsaAfphosphoro- aAfaG uAfcU thioates fGfGf fgUfc and a vinyl a c phosphateCfaGf cUfuU (VP) on uAfuU fuAfa the fcUfL Gfcsa antisense 96 sa strandAD- gscsu 696 usGfs 702 Ultra low 65698.1 uaaaa aauac fluorine ggdGaugucc content; cagu cUfuu no 2′F on (Ag ua the sense n)uuc agcsa strand,aL96 sa two 2′Fs on the antisense strand AD- gscsu 697 usdGs 703 No65699.1 uaaaa aauac fluorine ggdGa ugdTc content; cagu ccdTu no 2F′s(Agn) u on either uuc uaagc sense or aL96 sasa antisense strand AD-gscsu 698 usGfs 704 Low 65703.1 uaaAf aauAf fluorine aGfGf cUfGfcontent; Gfaca ucccU 10 total gu f 2′Fs on the auuca uUfua sense and L96agcsa antisense sa strands AD- gscsu 699 usGfs 705 Low 65704.1 uaaAfaauac fluorine aGfGf ugucc content; Gfaca cUfuu 6 total gu ua 2′Fs onauuca agcsa the sense L96 sa and antisense strands

The ability of the iRNAs to inhibit the expression of the human APOC3protein in vivo was tested in the APOC3-AAV mouse model system of APOC3overexpression. For a single dose screen study, APOC3-AAV micepreviously injected with 10¹¹ genome copies of APOC3 AAV wereadministered a single 3 mg/kg dose of AD-57553, AD-65696, AD-65703 andAD-65704 or PBS (as control), and the levels of APOC3 protein relativeto pre-dose were measured at days 0, 3, 10, 20, 30, 41, 55 and 70post-dose. FIG. 10 presents the data for all four tested iRNAs. The datain FIG. 10 indicate that iRNAs with low 2′F content (AD-65703 andAD-65704) are capable of sustaining an ˜80% of knock-down of APOC3 for30 days. The data also demonstrate that it takes 55-70 days for theAPOC3 levels to rebound to those observed in the control (PBS) groupfollowing a single 3 mg/kg dose.

AD-57553, AD-65696, AD-65699, AD-65703 and AD-65704 were used forfurther in vivo testing in dose response and multi-dose studies. For thedose response experiments, APOC3-AAV mice previously injected with 10¹¹genome copies of APOC3 AAV received four subcutaneous doses of 0.3mg/kg, 1 mg/kg and 3 mg/kg of each iRNA (Q2W×4 dosing schedule). Theanimals were bled at days 0, 7, 14, 21, 35, 49, 62 and 77, and theamount of APOC3 was evaluated. The dosing schedule used in theexperiment is depicted schematically in FIG. 11 .

The time courses for the 0.3 mg/kg, 1 mg/kg and 3 mg/kg doses are shownin FIGS. 12A, 12B and 12C, respectively. The data in FIGS. 12A-Cdemonstrate that at a 0.3 mg/kg dose each of the tested iRNAs is able toinhibit the expression of APOC3 protein up to 50% relative to pre-dosemeasurement. The data also demonstrate that at the 1 mg/kg and 3 mg/kgdoses, AD-65699, which contains no 2′F modifications, is less effectivethan the other tested iRNAs at inhibiting the expression of APOC3. Thedata further show that up to a 94% decrease in the relative APOC3 levelsis achieved after a 3 mg/kg dose of AD-57553, AD-65696, AD-65703 andAD-65704 is administered to the animals. This knock-down in the levelsof APOC3 is sustained for at least 3 weeks following the last dose. Twoof the tested iRNAs, AD-65696 and AD-65704 were able to sustain ˜80%inhibition of APOC3 protein for at least 5 weeks following the lastdose.

One of the selected lead sequences, AD-65704, containing 6 2′Fmodifications on the sense and antisense strand, was tested in asingle-dose titration study using the APOC3-AAV mouse model system ofAPOC3 overexpression. For dose screen experiments, APOC3-AAV micepreviously injected with 10¹¹ genome copies of hAPOC3 AAV wereadministered a single dose of 0.3 mg/kg, 1 mg/kg or 3 mg/kg of AD-65704or PBS (as control), and the levels of APOC3 protein relative topre-dose were measured after 14 days. The results, presented in FIG. 13, indicate that the dose effective to achieve 80% inhibition of theexpression of APOC3 (ED₈₀) is ˜3 mg/kg, while the dose effective toachieve 40% inhibition of the expression of APOC3 (ED₄₀) is 1 mg/kg.

For dose titration experiments, APOC3-AAV mice previously injected with10¹¹ genome copies of APOC3 AAV were subcutaneously administeredAD-65704 at a dose of 0.3 mg/kg, 1 mg/kg or 3 mg/kg. For each doselevel, a total of four doses were administered, one every other week(Q2W×4 administration). FIG. 14 shows the amount of APOC3 protein,relative to pre-dose, measured 20 days following the last dose. The dataindicate that the dose effective to achieve 90% inhibition of theexpression of APOC3 (ED₉₀) is ≤3 mg/kg; the dose effective to achieve70% inhibition of the expression of APOC3 (ED₇₀) is 1 mg/kg; and thedose effective to achieve 50% inhibition of the expression of APOC3(ED₈₀) is achieved at 0.3 mg/kg.

The results presented in this example (FIGS. 10-14 ) demonstrate thatiRNAs with low fluorine content (AD-65703 and AD-65704) achieve ˜80%knock-down of APOC3 that is sustained for at least 30 days whenadministered as a single 3 mg/kg dose (see FIG. 10 ). Administration offour 3 mg/kg doses every two weeks achieves up to 94% lowering of APOC3levels, with over 90% of knock-down sustained for at least 3 weeks (seeFIG. 12C).

The results for an iRNA with ultra low fluorine content (AD-65698)indicate that this iRNA is able to achieve up to 83% knock-down withfour doses of 3 mg/kg administered every two weeks, with 75% of theknock-down sustained for 3 weeks following the last dose (see FIG. 12C).The results for an iRNA with no fluorine content (AD-65699) indicatethat with a single dose of 3 mg/kg, this iRNA is able to achieve aknock-down that is very short-lived. Four 3 mg/kg doses administeredevery two weeks can increase the knock-down to up to 50%, with APOC3levels returning to baseline within 2 weeks after administration of thefinal dose (see FIG. 12C).

Addition of VP on the 5′ of the antisense strand results in a boost inthe ability of the iRNA to inhibit the expression of APOC3. This isevident from the 94% knock-down of APOC3 observed after a single 3 mg/kgdose of VP-containing AD-65696, as compared to ˜80% knock-down observedfor the parent iRNA AD-57553 without VP (see FIG. 10 ). Multi-dosingexperiments with AD-65696 resulted in up to 99% knock-down of APOC3 with3 mg/kg, ˜80% knock-down with 1 mg/kg and in >50% knock-down with 0.3mg/kg doses (see FIGS. 12A-C).

Example 8. Identification of iRNAs That Cross-React with Rabbit APOC3

The iRNA agents shown in Table 7 were analyzed for their ability tocross-react with rabbit APOC3. It was determined based on the number ofmismatches to rabbit APOC3 that AD-58911, AD-58924, AD-58922 andAD-58916 were cross-reactive with rabbit APOC3.

The four rabbit cross-reactive sequences were modified to contain atotal of 10 2′F modifications on the sense and antisense strandresulting in iRNAs AD-67221, AD-67222, AD-67223, and AD-67224. Theunmodified and modified sequences for AD-67221, AD-67222, AD-67223, andAD-67224 are presented, respectively, in Tables 11A and 11B below.

TABLE 11A Unmodified iRNAs That Cross-React to Rabbit APOC3. Sense SEQAntisense SEQ Duplex Sequence ID Sequence ID Name (5′ to 3′) NO:(5′ to 3′) NO: AD-67221.1 CCCAAUAA 706 UUCUUGUC 710 AGCUGGAC CAGCUUUAAAGAA UUGGGAG AD-67222.1 CCUCCCAA 707 UUGUCCAG 711 UAAAGCUG CUUUAUUGGACAA GGAGGCC AD-67223.1 UCCCAAUA 708 UCUUGUCC 712 AAGCUGGA AGCUUUAUCAAGA UGGGAGG AD-67224.1 CUCCCAAU 709 CUUGUCCA 713 AAAGCUGG GCUUUAUUACAAG GGGAGGC

TABLE 11B Modified iRNAs That Cross-React to Rabbit APOC3. Sense SEQAntisense SEQ Sequence ID Sequence ID Duplex Name (5′ to 3′) NO:(5′ to 3′) NO: AD-67221.1 cscscaau 714 usUfscuu 718 AfaAfGfC GfuCfCfafuggacaa gcuUfuAf gaaL96 uugggsas g AD-67222.1 cscsuccc 715 usUfsguc 719AfaUfAfA CfaGfCfu fagcugga uuaUfuGf caaL96 ggaggscs c AD-67223.1uscsccaa 716 usCfsuug 720 UfaAfAfG UfcCfAfg fcuggaca cuuUfaUf agaL96ugggasgs g AD-67224.1 csusccca 717 csUfsugu 721 AfuAfAfA CfcAfGfcfgcuggac uuuAfuUf aagL96 gggagsgs c

These iRNAs were tested in a single-dose study using the APOC3-AAV mousemodel system of APOC3 overexpression. APOC3-AAV mice previously injectedwith 10¹¹ genome copies of hAPOC3 AAV were administered a single dose of1 mg/kg of AD-65704, AD-67221, AD-67222, AD-67223, AD-67224, or PBS (ascontrol), and the levels of APOC3 protein relative to pre-dose weremeasured on days 14 and 26 post dose. The results, presented in FIG. 15, indicate that at day 14, iRNAs AD-67222 and AD-67224 were not activeat 1 mg/kg, AD-67223 showed 30% inhibition of APOC3 protein, andAD-67221 was comparable to AD-65704 with ˜40% inhibition of APOC3protein at 1 mg/kg dose. While initial silencing of APOC3 protein issimilar between AD-65704 and AD-67221 at day 14, the level of activityat day 26 indicates that AD-65704 is more durable than AD-67221,achieving 46% and 33% inhibition of APOC3 protein, respectively.

Example 9. Testing the Effect of Vinyl Phosphate Modification

The aim of this study was to test the effect of vinyl phosphate (VP) and2′F modifications on the antisense strand on the ability of iRNA toinhibit the expression of APOC3. The iRNAs used in the study aresummarized in Table 12 below.

TABLE 12 iRNAs used for the VP study Sense SEQ Antisense SEQ DuplexSequence ID Sequence ID Name (5′ to 3′) NO: (5′ to 3′) NO: AD-65698gscsuuaa 722 usGfsaau 728 aaggdGac acuguccc agu(Agn) Ufuuuaag uucaL96csasa AD-66239 gscsuuaa 723 VPusGfsa 729 aaggdGac AfuAfcUf agu(Agn)gUfcccUf uucaL96 uUfuAfaG fcsasa AD-65701 gscsuuaa 724 usGfsaau 730aaggdGac acuguccc agucuuca Ufuuuaag L96 csasa AD-66240 gscsuuaa 725VPusGfsa 731 aaggdGac AfuAfcUf agucuuca gUfcccUf L96 uUfuAfaG fcsasaAD-65704 gscsuuaa 726 usGfsaau 732 AfaGfGfG acuguccc facaguau UfuuuaagucaL96 csasa AD-66241 gscsuuaa 727 VPusGfsa 733 AfaGfGfG AfuAfcUffacaguau gUfcccUf ucaL96 uUfuAfaG fcsasa

iRNA agents AD-65698 and AD-66239 have the same sense strand butdifferent antisense strands. Similarly, duplexes AD-65701 and AD-66240have the same sense strand, but different antisense strands. iRNA agentsAD-65704 and AD-66241 contain the same sense strand, but differentantisense strands. The antisense strands for AD-65698, AD-65701 andAD-65704 are the same and contain four nucleotides containingphosphorothioates and two nucleotides containing a 2′-fluoromodification. The antisense strands for AD-66239, AD-66240 and AD-66241are the same and contain four nucleotides containing phosphorothioates,nine nucleotides containing a 2′-fluoro modification and one vinylphosphate at the 5′end of the antisense strand.

APOC3-AAV mice previously injected with 10¹¹ genome copies of APOC3 AAVwere administered a single 1 mg/kg dose of each iRNA listed in Table 11or PBS (as control), and the levels of APOC3 protein relative topre-dose were measured at days 10 and 24 post-dose. The data for day 10is shown in FIG. 16A, and the data for day 24 is shown in FIG. 16B.

The results demonstrate that mixing and matching of a VP modification onthe antisense strand with low fluorine content of the sense strandresulted in a similar boost in activity, however, was not able toimprove duration over pairing of the sense strand with antisense strandswithout the VP modification.

Example 10. In Vivo Testing of Additional iRNA Sequences Based onAD-65704

SAR optimization of AD-65704 chemistry generated 10 additional iRNAsequences (Table 13).

These iRNAs were tested in a single-dose study using the APOC3-AAV mousemodel system of APOC3 overexpression. Briefly, two weeks after injectingAPOC3-AAV mice with 10¹¹ genome copies of hAPOC3 AAV, the mice (n=3)were subcutaneously administered a single 1 mg/kg dose of the iRNAagents or PBS (as control). The serum levels of APOC3 protein relativeto pre-dose levels were measured by ELISA assay on days 14, 28, and 42post dose. The results, presented in FIGS. 17A and 17B, indicate that atday 14, all of the tested iRNAs were active at 1 mg/kg.

TABLE 13  Modified iRNA Sequences Sense SEQ Antisense SEQ DuplexSequence ID Sequence ID Name (5′ to 3′) NO: (5′ to 3′) NO: AD-65704.1gscsuuaa 734 usGfsaau 745 AfaGfGfG acuguccc facaguau Ufuuuaag ucaL96csasa AD-69528.1 gscsuuaa 735 usGfsaau 746 AfaGfGfG AfcUfguc facaguauccUfuUfu ucaL96 aagcsasa AD-69532.1 gscsuuaa 736 usGfsaau 747 AfaGfgGfacugucCf acaguauu cUfuuuaa caL96 gcsasa AD-69534.1 gscsuuaa 737 usGfsaau748 aaGfgGfa acuguccc caguauuc Ufuuuaag aL96 csasa AD-69535.1 gscsuuaa738 usGfsaau 749 aaGfgGfa acugucCf caguauuc cUfuuuaa aL96 gcsasaAD-69537.1 gscsuuaa 739 usGfsaau 750 aaGfgGfa acuguccc caguuuuc UfuuuaagaL96 csasa AD-69540.1 gscsuuaa 740 usGfsaau 751 aaGfgGfa acugucCfcaguuuuc cUfuuuaa aL96 gcsasa AD-69536.1 gscsuuaa 741 usGfsaau 752aaGfgGfa acuguccc cagudTuu Ufuuuaag caL96 csasa AD-69538.1 gscsuuaa 742usGfsaau 753 aaGfgGfa acuguccc cagu(Agn Ufuuuaag )uucaL96 csasaAD-69539.1 gscsuuaa 743 usGfsaau 754 aaGfgGfa acugucCf cagudTuu cUfuuuaacaL96 gcsasa AD-69541.1 gscsuuaa 744 usGfsaau 755 aaGfgGfa acugucCfcagu(Agn cUfuuuaa )uucaL96 gcsasa

Example 11. In Vivo Testing of iRNA Agents in Non-Human Primates

Based on the results described in Example 10, three iRNA agents,AD-65704, AD-69535 and AD-69541, were selected for evaluation innon-human primates.

Single and multi-dosing experiments were performed in Cynomolgusmonkeys. In one set of experiments, naïve male Cynomolgus monkeys (n=3)were subcutaneously administered a single weekly dose of 1 mg/kg dose ofAD-65704 on day 1, or naïve male Cynomolgus monkeys (n=3) weresubcutaneously administered a once weekly 1 mg/kg dose of AD-65704 ondays 1, 8, 15, 22, 29, 36, 43, and 50. Serum was collected on days −7,−1, 1, 8, 11, 15, 22, 29, 36, 43, 57, 64, and 71. The level ofCynomolgus ApoC3 protein was determined by ELISA. Liver biopsies wereperformed on days −7, 12, 30, and 64 and the level of ApoC3 mRNA wasdetermined. The results of the single-dose study are depreciated inFIGS. 18A and 18B and demonstrate that once weekly administration of 1mg/kg of AD-65704 achieves >80% lowering of total serum ApoC3 and up to50% lowering of total ApoC3 protein (18B). The data also demonstratethat once weekly administration of 1 mg/kg of AD-65704 achieves alowering of liver ApoC3 mRNA by 60% as compared to the predose level. Asdepicted in FIG. 18C, once weekly dosing of 1 mg/kg of AD-65704 lowersthe level of ApoC3 mRNA by 95% relative to the predose level.

In another set of experiments, naïve male Cynomolgus monkeys (n=3) weresubcutaneously administered a single weekly dose of 1 mg/kg dose ofAD-65704, AD-69535, or AD-69541 on day 1. Serum was collected on days−7, −1, 1, 8, 11, 15, 22, 29, and 36. The level of Cynomolgus ApoC3protein was determined by ELISA. Liver biopsies were performed on days−7, 12, 30, and 64 and the level of ApoC3 mRNA was determined. The datademonstrate that a single 1 mg/kg dose of all three of the iRNA agentstested lowers ApoC3 protein levels to about 50% of the baseline level(ED_(50 protein)=about 1 mg/kg) (19A). As demonstrated in FIG. 19B, asingle 1 mg/kg dose of AD-65704 lowers ApoC3 mRNA by 60% relative topre-dose levels, AD-69535 lowers ApoC3 mRNA by 68% relative to pre-doselevels, and AD-69541 lowers ApoC3 mRNA by 64% relative to pre-doselevels (ED_(50mRNA)<1 mg/kg).

An additional multi-dose study was performed in Cynomolgus monkeys withAD-65704, AD-69535, and AD-69541. Naïve male Cynomolgus monkeys weresubcutaneously administered a single 1 mg/kg dose AD-65704, AD-69535, orAD-69541 on day 1 and were subsequently administered a singlesubcutaneous 3 mg/kg dose of the same agent on day 36. N=3/group. Serumwas collected on days −7, −1, 1, 8, 11, 15, 22, 29, 36, 43, 50, 57, 64,and 71. The level of Cynomolgus ApoC3 protein was determined by ELISA.Liver biopsies were performed on days −7, 12, 30, and 64 and the levelof ApoC3 mRNA was determined. FIG. 20A demonstrates that administrationof a 3 mg/kg dose of AD-65704 or AD-69535 lowers the level of ApoC3protein by about 70% relative to pre-dose levels (ED_(70 protein)=about3 mg/kg) and FIG. 20B demonstrates that, at day 64 post-administrationof a 3 mg/kg dose of AD-65704, the level of ApoC3 mRNA is lowered by 81%relative to pre-dose levels, that at day 64 post-administration of a 3mg/kg dose of AD-69535, the level of ApoC3 mRNA is lowered by 88%relative to pre-dose levels, and that at day 64 post-administration of a3 mg/kg dose of AD-69541 the level of ApoC3 mRNA is lowered by 84%relative to pre-dose levels (ED_(80 mRNA)≤3 mg/kg).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A double stranded RNAi agent for inhibiting expression ofapolipoprotein C3 (APOC3) in a cell, wherein said double stranded RNAiagent comprises a sense strand and an antisense strand forming adouble-stranded region, wherein said sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:1, and said antisense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2, whereinsubstantially all of the nucleotides of at least one strand comprise anucleotide modification, and wherein said sense strand is conjugated toa ligand attached at the 3′-terminus.
 2. The double stranded RNAi agentof claim 1, wherein said antisense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense strand nucleotide sequences listed in Tables 4A,4B, 5, 8, 9, 10, 11A, 11B, and 12,
 13. 3. The double stranded RNAi agentof claim 1, wherein at least one of said nucleotide modifications isselected from the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide modification, a 2′-O-methyl modification, a 2′-fluoromodification, a 2′-deoxy-modification, a locked nucleotide modification,an unlocked nucleotide, a conformationally restricted nucleotidemodification, a constrained ethyl nucleotide modification, an abasicnucleotide modification, a 2′-amino-modified nucleotide modification, a2′-O-allyl-modification, 2′-C-alkyl modification,2′-hydroxyl-modification, a 2′-methoxyethyl modification, a 2′-O-alkylmodification, a morpholino nucleotide modification, a phosphoramidate,modification a non-natural base comprising nucleotide modification, atetrahydropyran modification, a 1,5-anhydrohexitol modification, acyclohexenyl modification, a nucleotide comprising a 5′-phosphorothioategroup modification, a nucleotide comprising a 5′-methylphosphonate groupmodification, a nucleotide comprising a 5′ phosphate or 5′ phosphatemimic modification, a nucleotide comprising vinyl phosphatemodification, a nucleotide comprising adenosine-glycol nucleic acid(GNA) modification, a nucleotide comprising thymidine-glycol nucleicacid (GNA) S-Isomer modification, a nucleotide comprising2-hydroxymethyl-tetrahydrofurane-5-phosphate modification, a nucleotidecomprising 2′-deoxythymidine-3′phosphate modification, a nucleotidecomprising 2′-deoxyguanosine-3′-phosphate modification, and a terminalnucleotide linked to a cholesteryl derivative or a dodecanoic acidbisdecylamide group modification.
 4. (canceled)
 5. The double strandedRNAi agent of claim 1, wherein all of the nucleotides of said sensestrand comprise a nucleotide modification; all of the nucleotides ofsaid antisense strand comprise a nucleotide modification; or all of thenucleotides of said sense strand and all of the nucleotides of saidantisense strand comprise a nucleotide modification.
 6. The doublestranded RNAi agent of claim 1, wherein at least one strand comprises a3′ overhang of at least 1 nucleotide; or at least one strand comprises a3′ overhang of at least 2 nucleotides.
 7. (canceled)
 8. (canceled) 9.The double stranded RNAi agent of claim 1, wherein the double-strandedregion is 15-30 nucleotide pairs in length; 17-23 nucleotide pairs inlength; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs inlength; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs inlength.
 10. The double stranded RNAi agent of claim 1, wherein eachstrand is independently has 15-30 nucleotides in length; or 19-30nucleotides in length.
 11. The double stranded RNAi agent of claim 7,wherein the nucleotide modifications are 2′-O-methyl modifications and2′-fluoro modifications.
 12. The double stranded RNAi agent of claim 1,wherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.
 13. The double stranded RNAiagent of claim 1, wherein the ligand is


14. The double stranded RNAi agent of claim 1, wherein the ligand isattached to the 3′ end of the sense strand.
 15. The double stranded RNAiagent of claim 14, wherein the double stranded RNAi agent is conjugatedto the ligand as shown in the following schematic

wherein X is O or S.
 16. The double stranded RNAi agent of claim 1,wherein said RNAi agent further comprises at least one phosphorothioateor methylphosphonate internucleotide linkage.
 17. A cell containing thedouble stranded RNAi agent of claim
 1. 18. A pharmaceutical compositioncomprising the double stranded RNAi agent of claim
 1. 19. (canceled) 20.The pharmaceutical composition of claim 18, wherein the double strandedRNAi agent is present in an unbuffered solution; or a buffered solution.21. A method of inhibiting apolipoprotein C3 (APOC3) expression in acell, the method comprising: (a) contacting the cell with the doublestranded RNAi agent of claim 1; and (b) maintaining the cell produced instep (a) for a time sufficient to obtain degradation of the mRNAtranscript of a APOC3 gene, thereby inhibiting expression of the APOC3gene in the cell.
 22. (canceled)
 23. A method of treating a subjecthaving an apolipoprotein C3 (APOC3) associated disease, comprisingadministering to the subject a therapeutically effective amount of thedouble stranded RNAi agent of claim 1, thereby treating said subject.24. The method of claim 23, wherein the APOC3 associated disease isselected from the group consisting of hypertriglyceridemia,non-alcoholic fatty liver disease, non-alcoholic steatohepatitis,polycystic ovary syndrome, kidney disease, obesity, type 2 diabetesmellitus (insulin resistance), hypertension, atherosclerosis andpancreatitis.
 25. (canceled)
 26. (canceled)